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Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter
ANALYTICALBIOCHEMISTRY124,
396-405 (1982)
Electrophoretic Transfer of Proteins from Sodium Dodecyl Polyacrylamide Gels to a Positively Charged Membrane JONATHAN M. GERSHONI' AND GEORGE
SulfateFilter
E. PALADE
Yale University School of Medicine, Section of Cell Biology, 333 Cedar Street, P.O. Box 3333, New Haven, Connecticut 06510 Received February 8, 1982 Zeta-bind, a positively charged nylon membrane, was tested as an immobilizing matrix for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels. It was found that Zeta-bind has a considerably greater capacity than does nitrocellulose for protein binding. Because of this property, more efficient elution of proteins from gels can be used (by omitting methanol from transfer buffers). The procedure described is more amenable to quantitation than usual nitrocellulose-based transfer. Antibody or lectin overlay techniques are also more sensitive on Zeta-bind than on nitrocellulose.
During the past 2 years numerous reports have described the transfer of proteins from SDS’-polyacrylamide gel electrophoretograms to either NC (l-6) or diazotized paper (2,7-9). When these transferred electrophoretograms (referred to hereafter as “transfers”) are reacted with antibodies (1,2,4,5,7-9), DNA, RNA (3), or lectins (6), it becomes possible to ascribe binding activities to specific polypeptide bands. Although overlay techniques can be used directly on gels (lo), transferring electrophoretograms to filters has the following advantages: (i) wet filters are pliable and easy to handle; (ii) the immobilized proteins are readily and equally accessible to various ligands (since the limitations introduced in gels by differential porosity are obviated); (iii) transfer analysis generally calls for small amounts of reagents; and (iv) processing times (incubations and washings) are significantly reduced. Regardless of whether transfer is mediated by passive diffusion (3), “blotting” (7), or electrophoresis ( 1,2,4-6, ’ To whom correspondence should be addressed. 2 Abbreviations used: SDS, sodium dodecyl sulfate; NC, nitrocellulose; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ZB, Zeta-bind. 0003-2697/82/120396-10$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
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8,9), it is still largely unknown whether the pattern of the resulting transfer is a quantitative or at least fully qualitative replica of the original electrophoretogram. In the case of electrophoretic transfer of proteins from SDS-polyacrylamide gel electrophoretograms, it was found by others (1,4) and by us that the elution of various proteins from gels as well as their subsequent adsorbance to NC can be differential. Under the conditions currently used, overlay assays on transfers obtained from the electrophoretograms yield essentially qualitative information. In this report we describe results obtained by transferring SDS-polyacrylamide gel electrophoretograms to a new filter ‘material on which overlay techniques are more sensitive and more amenable to quantitation than on NC. MATERIALS
AND METHODS
Materials. Nitrocellulose membrane filters (BA 85) were purchased from Schleicher and Schuell, Keene, New Hampshire. Zetabind (ZB) membrane filters were a gift from AMF, Specialty Materials Group/CUNO Division, Meriden, Connecticut. Stuphylococcus aureus protein A and concanavalian
ELECTROPHORETIC
TRANSFER
A were purchased from Pharmacia Fine Chemicals, Piscataway, New Jersey, and Calbiochem-Behring Corporation, La Jolla, California, respectively. BSA (A-7888), hemoglobin (H-2500), carbonic anhydrase, fetuin, ovalbumin, phosphorylase b, and soybean trypsin inhibitor were products of Sigma Chemical Company, St. Louis, Missouri. Carrier-free Na1251 was purchased from Amersham, Arlington Heights, Illinois. All the other reagents used were of analytical grade. Preparation of protein samples. Erythrocyte ghosts were prepared as previously described (11) from blood accumulated in the pleural cavity upon cardiac puncture of anesthesized CD1 mice. Bovine brain cortex homogenates were prepared as previously described (12). The radioiodination of protein standards, protein A, and concanavalin A was performed with 1,3,4,6-tetrachloro3a,6a-diphenylglycoluril (Iodo-Gen, Pierce Chemical Co., Rockford, Ill.) as described ( 13), except that the proteins were dissolved in PBS (1 mg/ml). Free “‘1 was separated from the ‘251-labeled proteins on either a 5-ml Bio-Gel P-2 column (Bio-Rad Laboratories, Richmond, Calif.) or on a 0.4-ml AG- 1-X8 column ( Bio-Rad Laboratories, Richmond, Calif.). The specific activities routinely obtained ranged from lo6 to lo7 cpm pg-’ protein. Polyacrylamide gel electrophoresis. Protein samples were solubilized in buffer containing (final concentrations) 2% (w/v) SDS, 2% (v/v) P-mercaptoethanol, 10% (v/v) glycerol, 0.1% (w/v) bromphenol blue, and 100 mM Tris-HCl, pH 6.8. The samples were boiled in this mixture for 3 min and then resolved, using Laemmli’s system (14) on 10% polyacrylamide slab gels. Electrophoretic transfer. Upon completion of electrophoresis, the gel (or portions of it) was placed on a wet Scotch-Brite pad (3M, St. Paul, Minn.) and its surface was rinsed with cool (8- 1O’C) transfer buffer (15.6 mM Tris-120 mM glycine, pH 8.3, with or without 20% (v/v) methanol). Tris (41
OF PROTEINS
397
mM)-boric acid (40 KIM), pH 8.3, was also tested. No significant differences were detected between the two buffer systems. A ZB or NC filter was then wetted by floating it on transfer buffer and placed on the gel, making sure that no air bubbles were caught within the filter or between the latter and the gel. The filter was then covered by a second Scotch-Brite pad. A number of such gel-filter assemblies separated by ScotchBrite pads can be mounted in sequence for simultaneous transfers. The assembly was placed between plastic grids, which were then snugly inserted-with the filter toward the anode-into a Plexiglas tank containing 4 liters of cool transfer buffer. Platinum electrodes in a configuration similar to that described by Bittner et al. (2) were secured to the wide walls of the tank. With an 8-cm distance between the anode and the cathode, the electric field generated during electrophoretic transfer appeared to be homogeneous. During transfer the buffer composition changed as salts were eluted from the gels, and thus the current increased when the voltage was kept constant. Because a standard power supply was used (Buchler Model 3-1500), it was sometimes found more practical to transfer at constant current (200 mA) and let the voltage gradually decrease. In a typical 2-h transfer, the voltage dropped, for instance, from 42 to 30 V. The voltage change could be avoided by prior equilibration of the gels with transfer buffer. This procedure could also prevent gel swelling during transfer, a common occurrence at acrylamide concentrations r 10%. The filters could be used immediately after transfer, or dried and stored between sheets of Whatman 3MM chromatography paper. In experiments in which transfer of “‘I-labeled protein was quantitated, both gels and filters were autoradiographed posttransfer using Kodak XAR-5 film and a DuPont Cronex Lightning Plus intensifying screen at -70°C. The radioactive bands were excised from gels and filters and counted in a Beckman Biogamma II counter.
398
GERSHONI
Staining of transfers. NC transfers were stained (N 1 min) with 0.1% (w/v) amido black 10B in 25% (v/v) isopropanol-10% (v/v) acetic acid in water, and destained ( -30 min) in 25% isopropanol- 10% acetic acid. Isopropanol was preferred over methanol because NC is less stable in the latter. Although Coomassie brilliant blue may also be used (4), a lower background was obtained when filters were stained with amido black 10B. To reduce the extent of shrinkage upon drying, stained filters were washed at length in PBS or water immediately after destaining and only then allowed to dry. Staining of ZB transfers presents a problem. Most of the dyes routinely used as protein stains are anions and bind tenaciously to ZB. Detergents (e.g., SDS, dodecyl trimethylammonium bromide, or Triton X100) at low concentrations (0.1% in water) remove the dyes from ZB, but at the same time destain the transferred proteins, SDS being the most effective destainer. A wide variety of cationic dyes was also tested and found not to bind readily to either ZB or transferred proteins. The silver stains tried (15-l 8) gave variable results and usually unacceptably high background (the most promising silver stain seemed to be that described in Ref. (15)). A number of histochemical protein stains were tested, and the Millon reaction for tyrosine (19) gave encouraging but insufficiently sensitive results. Of all the approaches tried, the best results were obtained using the following method. ZB transfers were briefly washed in 25% isopropanol- 10% acetic acid and then extensively washed in water. Next, they were incubated for 0.5 to 1 min in 0.1% (w/v) ferric chloride in water, washed in water, and incubated in 1% (w/v) tannic acid in water until a purple pattern appeared. The intensity of the stain was usually weak, therefore, this procedure requires improvement. Treatment of transfers with antibodies and lectins. To prevent nonspecific background binding, the filters must be quenched. For NC it was sufficient to incubate the fil-
AND PALADE
ters at room temperature for 1 h in 10 mM PBS, pH 7.4, containing either 2% (w/v) BSA or 1% (w/v) hemoglobin. Incubation of ZB filters in PBS, containing either 10% BSA or 1% hemoglobin, for at least 12 h at 45-50°C was necessary for satisfactory quenching. The quenched filters were reacted with the relevant ligands (e.g., antibodies, protein A, lectins) for 1 h at room temperature in PBS containing either 2% BSA or 1% hemoglobin and then washed at least 5 times in 50 to 100 ml PBS (20 min each wash). All solutions used in the overlay procedure contained sodium azide (0.05% w/v). The washed filters were autoradiographed at -70°C as described above. RESULTS
Binding
of Protein
to Filters
Under conditions currently used in electrophoretic transfer, most polypeptides are eluted from SDS-polyacrylamide electrophoretograms as anions. Therefore, the adsorbance of these polypeptides to NC (a negatively charged medium at pH 8) is not simply electrostatic but must involve other interactions, e.g., hydrophobic (20-22). We assumed that (by comparison with NC) a positively charged membrane material should have an appreciably higher affinity for negatively charged proteins. Such a positively charged filter, Zeta-bind, has recently been developed. ZB is a nylon-66-based membrane which, unlike other available nylon membranes (e.g., Gene-Screen-NEN, Boston, Mass.), has been modified by extensive cationization. Preliminary binding experiments were performed to compare the adsorbance of ‘2SI-labeled BSA to NC and ZB. Squares of filter material were incubated for 1 h in solutions of variable concentrations of “‘I-labeled BSA (O.Ol-5% w/v in PBS). After incubation, the filters were washed 5 times in PBS and then counted. The counts were used to calculate the amount of BSA bound per square centimeter (Fig. 1). The capacity
ELECTROPHORETIC
TRANSFER
FIG. 1. Binding of ?-labeled BSA to NC or ZB. Squares (4 cm’) of either NC (0) or ZB (0) were individually incubated for 1 h at room temperature in 3 ml of PBS in which BSA was dissolved at increasing concentrations (as shown). Each sample of the incubation medium contained an equal tracer amount of lz51-labeled BSA (177,000 f 6100 cpm ml-‘; estimated amount 180 ng ml-‘). After incubation, the filters were washed 5 times with 5 ml PBS and counted. The counts were converted to bound BSA (pg cm-*).
of ZB for binding BSA was consistently higher than that of NC. This was particularly evident when high concentrations of BSA were used; at 5% BSA, for instance, ZB and NC bound 480 fig cme2 and 80 pg cme2, respectively. Electrophoretic Transfer of Proteins from Gels to Filters Surprisingly, when ZB was tested in an actual electrophoretic transfer (1 h at 30 V) of 1251-labeled BSA (-200 ng), using buffer conditions similar to those described by Towbin et al. (i.e., 15.6 mM Tris, 120 mM glycine, 20% (v/v) methanol, pH 8.3), ZB showed no obvious advantage over NC, In such an experiment, 30% of the protein loaded could be eluted from the gels and was recovered on the corresponding ZB or NC filters. If the amount of protein eluted from the gel and subsequently available for binding were the limiting factor, the binding capacities of the two filters may not be suffi-
399
OF PROTEINS
ciently different for detection at low protein concentrations (see Fig. 1). To exploit the higher binding capacity of ZB, we tried to increase the efficiency of protein elution from the gel. The omission of methanol from the transfer buffer had such an effect: elution increased to >60 and -50% of the load when ZB and NC were used, respectively. In the absence of methanol, however, “‘Ilabeled BSA passed through at least five layers of NC filter (on which it was detected in decreasing amounts, e.g., 15 to 8% of the load); whereas most ‘251-labeled BSA (>60% of the load) could be retained on the first ZB filter, with ~1% detected on each sequential filter. Similar results were obtained with other protein standards (phosphorylase b, fetuin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor). A detailed analysis of a representative experiment in which ‘251-labeled BSA was transferred in the absence of methanol is presented in Figs. 2-4. They show that (i) more than 80% of the protein loaded on the gel could be accounted for when one or two layers of ZB were used (Figs. 2 and 3); (ii) 80% (at low load) to 50% (at high load) of the filter-bound BSA was recovered on the first layer of ZB (Fig. 4); and (iii) the use of more than three layers of ZB did not seem to improve the extent of recovery (Fig. 3). Similar results were not obtained even when 10 layers of NC were used (Figs. 2-4). Unaccounted 1251-labeled BSA (presumably lost in the buffer) amounted to >25% of the load when NC was used and to ~15% in the case of ZB. Retention Filters
of Transferred
Proteins
on
Once adsorbed to ZB, protein is very well retained, as demonstrated by the results of the following experiments. The ‘251-labeled BSA run on an SDS-polyacrylamide gel was electrophoretically transferred to either ZB or NC. After transfer, the filters were incubated in one of the following conditions: (i) 15 min in 0.5% glutaraldehyde in PBS, (ii) 1 h in 25% isopropanol-10% acetic acid,
400
GERSHONI
AND PALADE
60
70
60
retained ;; 50
tkr.
H F x 40 ,” m 730 2 t E .-5 20 ; 0 L s 6 IO
t
I
I
I
I
,,I,,,
I
I
106
I05 PII
BSA
loaded
(cpm)
FIG.2. Quantitation of transfer of variable amounts of ‘ZSI-labeled BSA to ZB or NC. Duplicates of four increasing concentrations of ‘z51-labeled BSA (estimated as 150, 300, 514, and 1400 ng) were run on 10% SDS-polyacrylamide gel. After electrophoresis, one series of lanes was transferred to eight sequential layers of ZB (0, n , A) and the other to 10 sequential layers of NC (0, 0, A). At the end of the transfer (2 h in Tris-boric acid, pH 8.3, without methanol, at constant 32 V), the gels and filters were counted. The amount of BSA eluted from each gel lane (0, 0) was calculated as the difference between radioactivity loaded and radioactivity remaining in the corresponding gel posttransfer. The unaccounted BSA (A, A) is the calculated difference between radioactivity eluted from each gel and radioactivity recovered on the corresponding ZB or NC filters (0, W).
and (iii) 1 h in PBS alone. Next the filters were rinsed a number of times in PBS and subsequently washed overnight in 0.1% Triton X-100 in PBS. The amount of label on each filter was determined after both transfer and detergent wash. In the case of NC it was found that 80% of the ‘2SI-labeled BSA was washed away from unfixed filters; although results were variable, at least 1.52 times more counts could be retained on such filters after the glutaraldehyde or the acidic-alcohol treatments. When ZB was used, >65% of the original counts were retained in the absence of any fixation and fixation increased this value to >90%. Fur-
thermore, the retention of protein to ZB seemed practically unaffected by variation in the pH (ranging from 2.0 to 8.3) of the washing solutions (Fig. 5). Overlaying
of Transfers
Before one can use a transferred pattern in any overlay technique, residual potential binding sites on the filter must be quenched to minimize nonspecific background. Here, the advantage of ZB, i.e., its high affinity for protein, becomes a point for concern. As shown in Fig. 1, 5% BSA in PBS does not saturate this filter material. It was found
ELECTROPHORETIC
TRANSFER
, I
2
3
4 Layer
5
6 of
7
6
9
IO
filter
FIG. 3. Recovery of ‘251-labeled BSA on eight and 10 successive layers of ZB and NC, respectively. Recovery of 12rI-labeled BSA on eight ZB filters (0) or 10 NC filters (0) from the maximal load of BSA (1400 ng) used. Arrow 1 indicates the total amount recovered on eight layers of ZB. Arrow 2 indicates the total amount recovered on 10 layers of NC.
that ZB could be quenched effectively when incubated in 10% BSA in PBS overnight at 4550°C. Lower temperature (e.g., 37”(J), lower concentrations of BSA, or shorter incubations of the filter with the above solution at 50°C resulted in unacceptably high background in overlays. Hemoglobin (1% in PBS at 45-50°C) was also found to be effective for quenching ZB transfers. The choice of the protein to be used as a quenching reagent depends on the type of overlay intended. As BSA may have contaminants that interfere with lectin overlays (6), hemoglobin has been used in such cases. On the other hand, although hemoglobin is usable for antibody overlays, its peroxidase
401
OF PROTEINS
activity would be detrimental were the antibody complexes to be detected enzymatitally with horseradish peroxidase conjugates. Overlays of protein patterns transferred to ZB or NC with antibodies or lectins are demonstrated in Figs. 6 and 7. It is apparent that the greater binding capacity of ZB over NC renders these techniques more sensitive when ZB is used. The specificity of lectin binding was tested by using appropriate haptens. In the case of lanes D and E in Fig. 7, for instance, the net radioactivity bound to the respective filters was determined by deducting from the total counts the background counts measured on the lower parts of the lanes. Net signal on ZB was 1.6 times higher than on NC. A first wash in PBS containing 100 mM a-methylglucoside removed 82 and 76% of the signal from ZB and NC, respectively. A second wash in PBS containing 100 mM a-methylmannoside increased the removal to 90% for ZB and 84% for NC.
100 so
16
30 ESA
Loaded
5.2 (IOscpm)
13.9
FIG. 4. Comparison of BSA recovery on the first ZB filter, the first NC filter, and 10 successive NC filters. The results obtained in the experiment presented in Fig. 2 were normalized to the amount of BSA recovered on all eight filters of ZB for each BSA concentration. This value is taken as 100% recovery. Note that for the three lower concentrations of BSA the first ZB filter (stippled bars) adsorbed as much as or more than all 10 layers of NC (hatched bars). In all cases the amount of BSA recovered on the first ZB filter was considerably greater (-4 times) than that recovered on the first NC filter (solid bars).
GERSHONI
402
AND PALADE
P-
F-
C-
S-
2.0 3.0 4.0 5.0 6.0 8.3 PH FIG. 5. The effect of pH on the retention of *251-labeled protein standards on ZB. Six aliquots of a mixture of 1251-labeled phosphorylase b (P), ‘2SI-labeled fetuin (F), ‘251-labeled carbonic anhydrase (C), and ‘251-labeled soybean trypsin inhibitor (S) were run on a 10% SDS-polyacrylamide gel. After electrophoresis, the lanes were separated and transferred to ZB in Tris-glycine buffer, pH 8.3, for 2 h at constant current (200 mA). The filters were then washed overnight in either 50 mM phosphate-citrate buffer at the pH indicated under each lane or in KCl-HCl buffer, pH 2.0, or not washed at all (pH 8.3). Each filter was autoradiographed for 12 h as described under Materials and Methods. The autoradiograms indicate that comparable amounts of radioactivity were retained in each band, irrespective of the filter treatment prior to autoradiography. Some loss of phosphorylase b might have occurred in the pH 2.0 wash.
As already noted by others, transfer efficiency is lower for high M, proteins (Figs. 6 and 7). DISCUSSION
Overlay techniques on protein transfers have expanded the usefulness of SDS-polyacrylamide gel electrophoresis. Although one can directly overlay gels, the transfer of electrophoretograms to filters quite often increases the efficiency of these procedures. Easier, uniform accessibility to reagents and
shorter processing times in the case of filters offer additional advantages. The transferred electrophoretograms can be accumulated and stored dry between sheets of blotting paper (Whatman 3MM) for as long as 1 year before being used for overlaying. The ‘251-labeled lectin overlays have been found to be extremely convenient, especially‘ because one can directly characterize the complex obtained by competing off the lectin with the relevant monosacharide hapten. This can be done even after autoradiography of the transfer. Repeated use of this type is
ELECTROPHORETIC
TRANSFER
CDE FIG. 6. Transfers of bovine brain cortex homogenates overlayed with anti-protein I (Synapsin). Aliquots of bovine brain cortex homogenates (25 pg each) were resolved on a 10% SDS-polyacrylamide gel and electrophoretically transferred to either NC or ZB filters over 2 h in Tris-glycine buffer at 200 mA. Lane A is the original electrophoretogram stained with Coomassie brilliant blue. Lane B is a Coomassie brilliant blue stained electrophoretogram after transfer to NC. Lane C is the corresponding NC transfer stained with amido black 10B. Lanes D and E are autoradiograms of transfers to NC and ZB, respectively, after being quenched with BSA and overlayed 1 h with dilute (1:300) rabbit serum containing anti-protein I (synapsin (12)). washed, and subsequently incubated with ‘*‘I-labeled protein A ( IO6 cpm total) for 1 h, and washed again as described under Materials and Methods. Note that the minor component of protein I is apparent in the ZB transfer but not in the NC transfer. The minor component can be demonstrated on NC filters when longer incubation times or higher concentration of antibody are used.
OF PROTEINS
403
not possible in the case of dried gels. Moreover, glycoproteins can be modified by treating the corresponding transfers with glycosidases prior to lectin overlaying. For instance, Ricinus communis lectin binding to murine erythrocyte gp 3 is enhanced by prior treatment with neuraminidase (unpublished data). The transfers of preparative slab gels (i.e., a gel on which one sample is applied along the whole width of the gel) were also found useful in the screening of monoclonal antibodies. A transfer may be cut into thin (5 mm) longitudinal strips, and each strip can be used individually for antibody overlays. With narrow filter strips (that still represent the complete protein pattern of interest), the volume of reagents required are greatly reduced. In fact, by placing such a filter strip along the diagonal of a rectangular piece of nylon screening (3 X 20 cm) and by rolling up the assembly along the length of the rectangle, a well-supported filter strip of helical configuration (3 cm high) is obtained. This helical filter strip can be placed in a small scintillation vial and reacted (under rotation) with as little as 2 ml of reagent. In this manner a large number of strips can be processed simultaneously. To improve the transfer technique, we have tried to make it more sensitive and more amenable to quantitation than is possible by current procedures. This intent demanded finding conditions for efficient elution of proteins from gels and maximal retention of transferred protein on filters. The electrophoretic elution of proteins from SDS-polyacryalmide gel electrophoretograms in buffer containing methanol has been shown to be efficient (>90% recovery) only when electrophoresis is carried out for 16 to 22 h (4). We have shown that the omission of methanol from the transfer buffer increases the elution efficiency of proteins from SDS-polyacrylamide gel. However, under such conditions the protein-binding capacity of NC is greatly exceeded. Within
404
GERSHONI
15 min of electrophoretic transfer of ‘2sI-labeled BSA in the absence of methanol, protein can be detected on three sequential layers of NC. After 1 h, - 10% of the protein eluted could be found on the last of the five sequential layers of NC, and after 2 h of transfer, protein was shown to flow through 10 NC layers (Fig. 3). Under the same transfer conditions, ZB was found to be considerably more effective than NC as an immobilizing matrix: in seven experiments carried out in parallel, 72.15% f 4.73 (SE) of the protein eluted from each gel (as defined in Fig. 3) was recovered on the first ZB filter, as opposed to 20.35% f 2.24 (SE) found on the first NC filter. On account of the highbinding capacity of ZB for proteins, the rate (and extent) of protein elution from gels can be increased, the time needed for transfer can be greatly reduced, and larger loads of proteins can be handled. Moreover, as the retention of transferred proteins on ZB is superior to that on NC, higher signals are obtained rendering the overlay techniques more sensitive (Figs. 6 and 7). Due to its high affinity for anions, ZB may prove to be extremely useful for RNA and DNA gel analyses. Indeed, preliminary results show that [32P]RNA binds well to ZB. The high binding capacity of this material has, however, some disadvantages. Relatively long treatment (12 h) at high temperature (45-50°C) with either 10% BSA or 1% hemoglobin is required to insure effective quenching of unbound sites. These conditions should not be detrimental for most proteins. In fact, most often protein samples are boiled prior to electrophoresis. In addition, protein patterns on ZB filters cannot be easily stained because most dyes commonly used as protein stains are themselves anionic (e.g., Coomassie brilliant blue or amido black 10B) and bind to the filter. As yet no simple method for staining proteins on ZB has been found, although many dyes, reagents, and different approaches have been tried (see Materials and Methods).
AND
PALADE
ABCDEFG FIG. 7. ‘*sI-labeled lectin overlay of electrophoretic transfers from SDS-polyacrylamide gel electrophoretograms of murine erythrocytic ghosts. Aliquots of murine erythrocytic ghosts (25 pg each) were resolved on a 10% SDS-polyacrylamide gel and electrophoretically transferred to either NC or ZB filters for 2 h in Trisglycine buffer at 200 mA. Lane A is the original electrophoretogram stained with Coomassie brilliant blue. Lane B is a Coomassie brilliant blue stained electrophoretogram after transfer to an NC filter. Lane C is the corresponding NC transfer stained with amido black 10B. Lane D is an autoradiogram of a hemoglobinquenched NC transfer overlayed 1 h with ‘251-labeled concanavilin A (5 X 10’ cpm total) and washed as described under Materials and Methods. Lane F is the same filter after washing in succession with a-methylglucoside (100 mM in PBS) and cy-methylmannoside (100 mM in PBS). Lanes E and G are ZB transfers treated in the same way as lanes D and F, respectively.
ELECTROPHORETIC
TRANSFER
In conclusion, ZB appears to be more useful than other available filter materials for SDS-polyacrylamide gel electrophoretogram transfers and subsequent overlay techniques, because of its high and tenacious binding capacity for proteins. Binding properties of this type are of critical importance in transfer technology.
ACKNOWLEDGMENTS We thank Dr. Pietro DeCamilli, Ms. Penny Miller, and Dr. Carl Roman0 for providing samples of bovine brain homogenates and anti-protein I; Dr. Ira Mellman for useful advice “with protein radioiodination; Mrs. Cindy Davis for typing the manuscript; and Dr. Zaid Smith for sustained critical discussion. This work was supported by NIH Grant GM 27303 and by a Chaim Weizmann Postdoctoral Fellowship (J. M. G).
REFERENCES 1. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Nat. Acad. Sci. USA 76, 4350-4354. 2. Bittner, M., Kupferer, P., and Morris, C. (1980) Anal. Biochem. 102,459-471. 3. Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) Nucf. Acids Rex 8, I-20. 4. Burnette, W. N. (1981) Anal. Biochem. 112, 195203. 5.
Glass, W. F., Briggs, R. C., and Hnilica, L. S. (1981) Science 211, 70-72.
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6. Glass, W. F., Briggs, R. C., and Hnilica, L. S. (1981) Anal. Biochem. 115, 219-224. 7. Renart, J., Reiser, J., and Stark, G. R. (1979) Proc. Nat. Acad. Sci. USA 76, 3116-3120. 8. Stellwag, E. J., and Dahlberg, A. E. (1980) Nucl. Acids Res. 8, 299-3 17. 9. Symington, J., Green, M., and Bra&man, K. (1981) Proc. Nat. Acad. Sci. USA 78, 177-181. 10. Burridge, K. (1978) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 50, pp. 54-64, Academic Press, New York. 11. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617. 12. DeCamilli, P., Ueda, T., Bloom, F. E., Battenerg, E., and Greengard, P. (1979) Proc. Nat. Acad. Sci. USA 76, 5977-598 1. 13. Fraker, P. J., and Speck, J. C. (1978) Biochem. Biophys. Rex Commun. 80, 849-857. 14. Laemmli, I-l. K. (1970) Nature flondon) 227,680685.
15. Sammons, D. W., Adams, L. D., and Nishizawa, E. E. (1981) Electrophoresis 2, 135-141. 16. Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) Anal. Biochem. 98, 231-237. 17. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Eiochem. 105, 361-363. 18. Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1980) Science 211, 1437-1438. 19. Troyer, H. (1980) in Principles and Techniques of Histochemistry, 1st ed., Little, Brown, Boston. 20. Wallis, C., Melnick, J. L., and Gerba, C. P. (1979) Annu. Rev. Microbial. 33,413-437. 21. Schneider, Z. (1980) Anal. Biochem. 108,96-103. 22. Farrah, S. R., Shah, D. O., and Ingram, L. 0. (1981) Proc. Nat. Acad. Sci. USA 78, 12291232.
396-405 (1982)
Electrophoretic Transfer of Proteins from Sodium Dodecyl Polyacrylamide Gels to a Positively Charged Membrane JONATHAN M. GERSHONI' AND GEORGE
SulfateFilter
E. PALADE
Yale University School of Medicine, Section of Cell Biology, 333 Cedar Street, P.O. Box 3333, New Haven, Connecticut 06510 Received February 8, 1982 Zeta-bind, a positively charged nylon membrane, was tested as an immobilizing matrix for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels. It was found that Zeta-bind has a considerably greater capacity than does nitrocellulose for protein binding. Because of this property, more efficient elution of proteins from gels can be used (by omitting methanol from transfer buffers). The procedure described is more amenable to quantitation than usual nitrocellulose-based transfer. Antibody or lectin overlay techniques are also more sensitive on Zeta-bind than on nitrocellulose.
During the past 2 years numerous reports have described the transfer of proteins from SDS’-polyacrylamide gel electrophoretograms to either NC (l-6) or diazotized paper (2,7-9). When these transferred electrophoretograms (referred to hereafter as “transfers”) are reacted with antibodies (1,2,4,5,7-9), DNA, RNA (3), or lectins (6), it becomes possible to ascribe binding activities to specific polypeptide bands. Although overlay techniques can be used directly on gels (lo), transferring electrophoretograms to filters has the following advantages: (i) wet filters are pliable and easy to handle; (ii) the immobilized proteins are readily and equally accessible to various ligands (since the limitations introduced in gels by differential porosity are obviated); (iii) transfer analysis generally calls for small amounts of reagents; and (iv) processing times (incubations and washings) are significantly reduced. Regardless of whether transfer is mediated by passive diffusion (3), “blotting” (7), or electrophoresis ( 1,2,4-6, ’ To whom correspondence should be addressed. 2 Abbreviations used: SDS, sodium dodecyl sulfate; NC, nitrocellulose; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ZB, Zeta-bind. 0003-2697/82/120396-10$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
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8,9), it is still largely unknown whether the pattern of the resulting transfer is a quantitative or at least fully qualitative replica of the original electrophoretogram. In the case of electrophoretic transfer of proteins from SDS-polyacrylamide gel electrophoretograms, it was found by others (1,4) and by us that the elution of various proteins from gels as well as their subsequent adsorbance to NC can be differential. Under the conditions currently used, overlay assays on transfers obtained from the electrophoretograms yield essentially qualitative information. In this report we describe results obtained by transferring SDS-polyacrylamide gel electrophoretograms to a new filter ‘material on which overlay techniques are more sensitive and more amenable to quantitation than on NC. MATERIALS
AND METHODS
Materials. Nitrocellulose membrane filters (BA 85) were purchased from Schleicher and Schuell, Keene, New Hampshire. Zetabind (ZB) membrane filters were a gift from AMF, Specialty Materials Group/CUNO Division, Meriden, Connecticut. Stuphylococcus aureus protein A and concanavalian
ELECTROPHORETIC
TRANSFER
A were purchased from Pharmacia Fine Chemicals, Piscataway, New Jersey, and Calbiochem-Behring Corporation, La Jolla, California, respectively. BSA (A-7888), hemoglobin (H-2500), carbonic anhydrase, fetuin, ovalbumin, phosphorylase b, and soybean trypsin inhibitor were products of Sigma Chemical Company, St. Louis, Missouri. Carrier-free Na1251 was purchased from Amersham, Arlington Heights, Illinois. All the other reagents used were of analytical grade. Preparation of protein samples. Erythrocyte ghosts were prepared as previously described (11) from blood accumulated in the pleural cavity upon cardiac puncture of anesthesized CD1 mice. Bovine brain cortex homogenates were prepared as previously described (12). The radioiodination of protein standards, protein A, and concanavalin A was performed with 1,3,4,6-tetrachloro3a,6a-diphenylglycoluril (Iodo-Gen, Pierce Chemical Co., Rockford, Ill.) as described ( 13), except that the proteins were dissolved in PBS (1 mg/ml). Free “‘1 was separated from the ‘251-labeled proteins on either a 5-ml Bio-Gel P-2 column (Bio-Rad Laboratories, Richmond, Calif.) or on a 0.4-ml AG- 1-X8 column ( Bio-Rad Laboratories, Richmond, Calif.). The specific activities routinely obtained ranged from lo6 to lo7 cpm pg-’ protein. Polyacrylamide gel electrophoresis. Protein samples were solubilized in buffer containing (final concentrations) 2% (w/v) SDS, 2% (v/v) P-mercaptoethanol, 10% (v/v) glycerol, 0.1% (w/v) bromphenol blue, and 100 mM Tris-HCl, pH 6.8. The samples were boiled in this mixture for 3 min and then resolved, using Laemmli’s system (14) on 10% polyacrylamide slab gels. Electrophoretic transfer. Upon completion of electrophoresis, the gel (or portions of it) was placed on a wet Scotch-Brite pad (3M, St. Paul, Minn.) and its surface was rinsed with cool (8- 1O’C) transfer buffer (15.6 mM Tris-120 mM glycine, pH 8.3, with or without 20% (v/v) methanol). Tris (41
OF PROTEINS
397
mM)-boric acid (40 KIM), pH 8.3, was also tested. No significant differences were detected between the two buffer systems. A ZB or NC filter was then wetted by floating it on transfer buffer and placed on the gel, making sure that no air bubbles were caught within the filter or between the latter and the gel. The filter was then covered by a second Scotch-Brite pad. A number of such gel-filter assemblies separated by ScotchBrite pads can be mounted in sequence for simultaneous transfers. The assembly was placed between plastic grids, which were then snugly inserted-with the filter toward the anode-into a Plexiglas tank containing 4 liters of cool transfer buffer. Platinum electrodes in a configuration similar to that described by Bittner et al. (2) were secured to the wide walls of the tank. With an 8-cm distance between the anode and the cathode, the electric field generated during electrophoretic transfer appeared to be homogeneous. During transfer the buffer composition changed as salts were eluted from the gels, and thus the current increased when the voltage was kept constant. Because a standard power supply was used (Buchler Model 3-1500), it was sometimes found more practical to transfer at constant current (200 mA) and let the voltage gradually decrease. In a typical 2-h transfer, the voltage dropped, for instance, from 42 to 30 V. The voltage change could be avoided by prior equilibration of the gels with transfer buffer. This procedure could also prevent gel swelling during transfer, a common occurrence at acrylamide concentrations r 10%. The filters could be used immediately after transfer, or dried and stored between sheets of Whatman 3MM chromatography paper. In experiments in which transfer of “‘I-labeled protein was quantitated, both gels and filters were autoradiographed posttransfer using Kodak XAR-5 film and a DuPont Cronex Lightning Plus intensifying screen at -70°C. The radioactive bands were excised from gels and filters and counted in a Beckman Biogamma II counter.
398
GERSHONI
Staining of transfers. NC transfers were stained (N 1 min) with 0.1% (w/v) amido black 10B in 25% (v/v) isopropanol-10% (v/v) acetic acid in water, and destained ( -30 min) in 25% isopropanol- 10% acetic acid. Isopropanol was preferred over methanol because NC is less stable in the latter. Although Coomassie brilliant blue may also be used (4), a lower background was obtained when filters were stained with amido black 10B. To reduce the extent of shrinkage upon drying, stained filters were washed at length in PBS or water immediately after destaining and only then allowed to dry. Staining of ZB transfers presents a problem. Most of the dyes routinely used as protein stains are anions and bind tenaciously to ZB. Detergents (e.g., SDS, dodecyl trimethylammonium bromide, or Triton X100) at low concentrations (0.1% in water) remove the dyes from ZB, but at the same time destain the transferred proteins, SDS being the most effective destainer. A wide variety of cationic dyes was also tested and found not to bind readily to either ZB or transferred proteins. The silver stains tried (15-l 8) gave variable results and usually unacceptably high background (the most promising silver stain seemed to be that described in Ref. (15)). A number of histochemical protein stains were tested, and the Millon reaction for tyrosine (19) gave encouraging but insufficiently sensitive results. Of all the approaches tried, the best results were obtained using the following method. ZB transfers were briefly washed in 25% isopropanol- 10% acetic acid and then extensively washed in water. Next, they were incubated for 0.5 to 1 min in 0.1% (w/v) ferric chloride in water, washed in water, and incubated in 1% (w/v) tannic acid in water until a purple pattern appeared. The intensity of the stain was usually weak, therefore, this procedure requires improvement. Treatment of transfers with antibodies and lectins. To prevent nonspecific background binding, the filters must be quenched. For NC it was sufficient to incubate the fil-
AND PALADE
ters at room temperature for 1 h in 10 mM PBS, pH 7.4, containing either 2% (w/v) BSA or 1% (w/v) hemoglobin. Incubation of ZB filters in PBS, containing either 10% BSA or 1% hemoglobin, for at least 12 h at 45-50°C was necessary for satisfactory quenching. The quenched filters were reacted with the relevant ligands (e.g., antibodies, protein A, lectins) for 1 h at room temperature in PBS containing either 2% BSA or 1% hemoglobin and then washed at least 5 times in 50 to 100 ml PBS (20 min each wash). All solutions used in the overlay procedure contained sodium azide (0.05% w/v). The washed filters were autoradiographed at -70°C as described above. RESULTS
Binding
of Protein
to Filters
Under conditions currently used in electrophoretic transfer, most polypeptides are eluted from SDS-polyacrylamide electrophoretograms as anions. Therefore, the adsorbance of these polypeptides to NC (a negatively charged medium at pH 8) is not simply electrostatic but must involve other interactions, e.g., hydrophobic (20-22). We assumed that (by comparison with NC) a positively charged membrane material should have an appreciably higher affinity for negatively charged proteins. Such a positively charged filter, Zeta-bind, has recently been developed. ZB is a nylon-66-based membrane which, unlike other available nylon membranes (e.g., Gene-Screen-NEN, Boston, Mass.), has been modified by extensive cationization. Preliminary binding experiments were performed to compare the adsorbance of ‘2SI-labeled BSA to NC and ZB. Squares of filter material were incubated for 1 h in solutions of variable concentrations of “‘I-labeled BSA (O.Ol-5% w/v in PBS). After incubation, the filters were washed 5 times in PBS and then counted. The counts were used to calculate the amount of BSA bound per square centimeter (Fig. 1). The capacity
ELECTROPHORETIC
TRANSFER
FIG. 1. Binding of ?-labeled BSA to NC or ZB. Squares (4 cm’) of either NC (0) or ZB (0) were individually incubated for 1 h at room temperature in 3 ml of PBS in which BSA was dissolved at increasing concentrations (as shown). Each sample of the incubation medium contained an equal tracer amount of lz51-labeled BSA (177,000 f 6100 cpm ml-‘; estimated amount 180 ng ml-‘). After incubation, the filters were washed 5 times with 5 ml PBS and counted. The counts were converted to bound BSA (pg cm-*).
of ZB for binding BSA was consistently higher than that of NC. This was particularly evident when high concentrations of BSA were used; at 5% BSA, for instance, ZB and NC bound 480 fig cme2 and 80 pg cme2, respectively. Electrophoretic Transfer of Proteins from Gels to Filters Surprisingly, when ZB was tested in an actual electrophoretic transfer (1 h at 30 V) of 1251-labeled BSA (-200 ng), using buffer conditions similar to those described by Towbin et al. (i.e., 15.6 mM Tris, 120 mM glycine, 20% (v/v) methanol, pH 8.3), ZB showed no obvious advantage over NC, In such an experiment, 30% of the protein loaded could be eluted from the gels and was recovered on the corresponding ZB or NC filters. If the amount of protein eluted from the gel and subsequently available for binding were the limiting factor, the binding capacities of the two filters may not be suffi-
399
OF PROTEINS
ciently different for detection at low protein concentrations (see Fig. 1). To exploit the higher binding capacity of ZB, we tried to increase the efficiency of protein elution from the gel. The omission of methanol from the transfer buffer had such an effect: elution increased to >60 and -50% of the load when ZB and NC were used, respectively. In the absence of methanol, however, “‘Ilabeled BSA passed through at least five layers of NC filter (on which it was detected in decreasing amounts, e.g., 15 to 8% of the load); whereas most ‘251-labeled BSA (>60% of the load) could be retained on the first ZB filter, with ~1% detected on each sequential filter. Similar results were obtained with other protein standards (phosphorylase b, fetuin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor). A detailed analysis of a representative experiment in which ‘251-labeled BSA was transferred in the absence of methanol is presented in Figs. 2-4. They show that (i) more than 80% of the protein loaded on the gel could be accounted for when one or two layers of ZB were used (Figs. 2 and 3); (ii) 80% (at low load) to 50% (at high load) of the filter-bound BSA was recovered on the first layer of ZB (Fig. 4); and (iii) the use of more than three layers of ZB did not seem to improve the extent of recovery (Fig. 3). Similar results were not obtained even when 10 layers of NC were used (Figs. 2-4). Unaccounted 1251-labeled BSA (presumably lost in the buffer) amounted to >25% of the load when NC was used and to ~15% in the case of ZB. Retention Filters
of Transferred
Proteins
on
Once adsorbed to ZB, protein is very well retained, as demonstrated by the results of the following experiments. The ‘251-labeled BSA run on an SDS-polyacrylamide gel was electrophoretically transferred to either ZB or NC. After transfer, the filters were incubated in one of the following conditions: (i) 15 min in 0.5% glutaraldehyde in PBS, (ii) 1 h in 25% isopropanol-10% acetic acid,
400
GERSHONI
AND PALADE
60
70
60
retained ;; 50
tkr.
H F x 40 ,” m 730 2 t E .-5 20 ; 0 L s 6 IO
t
I
I
I
I
,,I,,,
I
I
106
I05 PII
BSA
loaded
(cpm)
FIG.2. Quantitation of transfer of variable amounts of ‘ZSI-labeled BSA to ZB or NC. Duplicates of four increasing concentrations of ‘z51-labeled BSA (estimated as 150, 300, 514, and 1400 ng) were run on 10% SDS-polyacrylamide gel. After electrophoresis, one series of lanes was transferred to eight sequential layers of ZB (0, n , A) and the other to 10 sequential layers of NC (0, 0, A). At the end of the transfer (2 h in Tris-boric acid, pH 8.3, without methanol, at constant 32 V), the gels and filters were counted. The amount of BSA eluted from each gel lane (0, 0) was calculated as the difference between radioactivity loaded and radioactivity remaining in the corresponding gel posttransfer. The unaccounted BSA (A, A) is the calculated difference between radioactivity eluted from each gel and radioactivity recovered on the corresponding ZB or NC filters (0, W).
and (iii) 1 h in PBS alone. Next the filters were rinsed a number of times in PBS and subsequently washed overnight in 0.1% Triton X-100 in PBS. The amount of label on each filter was determined after both transfer and detergent wash. In the case of NC it was found that 80% of the ‘2SI-labeled BSA was washed away from unfixed filters; although results were variable, at least 1.52 times more counts could be retained on such filters after the glutaraldehyde or the acidic-alcohol treatments. When ZB was used, >65% of the original counts were retained in the absence of any fixation and fixation increased this value to >90%. Fur-
thermore, the retention of protein to ZB seemed practically unaffected by variation in the pH (ranging from 2.0 to 8.3) of the washing solutions (Fig. 5). Overlaying
of Transfers
Before one can use a transferred pattern in any overlay technique, residual potential binding sites on the filter must be quenched to minimize nonspecific background. Here, the advantage of ZB, i.e., its high affinity for protein, becomes a point for concern. As shown in Fig. 1, 5% BSA in PBS does not saturate this filter material. It was found
ELECTROPHORETIC
TRANSFER
, I
2
3
4 Layer
5
6 of
7
6
9
IO
filter
FIG. 3. Recovery of ‘251-labeled BSA on eight and 10 successive layers of ZB and NC, respectively. Recovery of 12rI-labeled BSA on eight ZB filters (0) or 10 NC filters (0) from the maximal load of BSA (1400 ng) used. Arrow 1 indicates the total amount recovered on eight layers of ZB. Arrow 2 indicates the total amount recovered on 10 layers of NC.
that ZB could be quenched effectively when incubated in 10% BSA in PBS overnight at 4550°C. Lower temperature (e.g., 37”(J), lower concentrations of BSA, or shorter incubations of the filter with the above solution at 50°C resulted in unacceptably high background in overlays. Hemoglobin (1% in PBS at 45-50°C) was also found to be effective for quenching ZB transfers. The choice of the protein to be used as a quenching reagent depends on the type of overlay intended. As BSA may have contaminants that interfere with lectin overlays (6), hemoglobin has been used in such cases. On the other hand, although hemoglobin is usable for antibody overlays, its peroxidase
401
OF PROTEINS
activity would be detrimental were the antibody complexes to be detected enzymatitally with horseradish peroxidase conjugates. Overlays of protein patterns transferred to ZB or NC with antibodies or lectins are demonstrated in Figs. 6 and 7. It is apparent that the greater binding capacity of ZB over NC renders these techniques more sensitive when ZB is used. The specificity of lectin binding was tested by using appropriate haptens. In the case of lanes D and E in Fig. 7, for instance, the net radioactivity bound to the respective filters was determined by deducting from the total counts the background counts measured on the lower parts of the lanes. Net signal on ZB was 1.6 times higher than on NC. A first wash in PBS containing 100 mM a-methylglucoside removed 82 and 76% of the signal from ZB and NC, respectively. A second wash in PBS containing 100 mM a-methylmannoside increased the removal to 90% for ZB and 84% for NC.
100 so
16
30 ESA
Loaded
5.2 (IOscpm)
13.9
FIG. 4. Comparison of BSA recovery on the first ZB filter, the first NC filter, and 10 successive NC filters. The results obtained in the experiment presented in Fig. 2 were normalized to the amount of BSA recovered on all eight filters of ZB for each BSA concentration. This value is taken as 100% recovery. Note that for the three lower concentrations of BSA the first ZB filter (stippled bars) adsorbed as much as or more than all 10 layers of NC (hatched bars). In all cases the amount of BSA recovered on the first ZB filter was considerably greater (-4 times) than that recovered on the first NC filter (solid bars).
GERSHONI
402
AND PALADE
P-
F-
C-
S-
2.0 3.0 4.0 5.0 6.0 8.3 PH FIG. 5. The effect of pH on the retention of *251-labeled protein standards on ZB. Six aliquots of a mixture of 1251-labeled phosphorylase b (P), ‘2SI-labeled fetuin (F), ‘251-labeled carbonic anhydrase (C), and ‘251-labeled soybean trypsin inhibitor (S) were run on a 10% SDS-polyacrylamide gel. After electrophoresis, the lanes were separated and transferred to ZB in Tris-glycine buffer, pH 8.3, for 2 h at constant current (200 mA). The filters were then washed overnight in either 50 mM phosphate-citrate buffer at the pH indicated under each lane or in KCl-HCl buffer, pH 2.0, or not washed at all (pH 8.3). Each filter was autoradiographed for 12 h as described under Materials and Methods. The autoradiograms indicate that comparable amounts of radioactivity were retained in each band, irrespective of the filter treatment prior to autoradiography. Some loss of phosphorylase b might have occurred in the pH 2.0 wash.
As already noted by others, transfer efficiency is lower for high M, proteins (Figs. 6 and 7). DISCUSSION
Overlay techniques on protein transfers have expanded the usefulness of SDS-polyacrylamide gel electrophoresis. Although one can directly overlay gels, the transfer of electrophoretograms to filters quite often increases the efficiency of these procedures. Easier, uniform accessibility to reagents and
shorter processing times in the case of filters offer additional advantages. The transferred electrophoretograms can be accumulated and stored dry between sheets of blotting paper (Whatman 3MM) for as long as 1 year before being used for overlaying. The ‘251-labeled lectin overlays have been found to be extremely convenient, especially‘ because one can directly characterize the complex obtained by competing off the lectin with the relevant monosacharide hapten. This can be done even after autoradiography of the transfer. Repeated use of this type is
ELECTROPHORETIC
TRANSFER
CDE FIG. 6. Transfers of bovine brain cortex homogenates overlayed with anti-protein I (Synapsin). Aliquots of bovine brain cortex homogenates (25 pg each) were resolved on a 10% SDS-polyacrylamide gel and electrophoretically transferred to either NC or ZB filters over 2 h in Tris-glycine buffer at 200 mA. Lane A is the original electrophoretogram stained with Coomassie brilliant blue. Lane B is a Coomassie brilliant blue stained electrophoretogram after transfer to NC. Lane C is the corresponding NC transfer stained with amido black 10B. Lanes D and E are autoradiograms of transfers to NC and ZB, respectively, after being quenched with BSA and overlayed 1 h with dilute (1:300) rabbit serum containing anti-protein I (synapsin (12)). washed, and subsequently incubated with ‘*‘I-labeled protein A ( IO6 cpm total) for 1 h, and washed again as described under Materials and Methods. Note that the minor component of protein I is apparent in the ZB transfer but not in the NC transfer. The minor component can be demonstrated on NC filters when longer incubation times or higher concentration of antibody are used.
OF PROTEINS
403
not possible in the case of dried gels. Moreover, glycoproteins can be modified by treating the corresponding transfers with glycosidases prior to lectin overlaying. For instance, Ricinus communis lectin binding to murine erythrocyte gp 3 is enhanced by prior treatment with neuraminidase (unpublished data). The transfers of preparative slab gels (i.e., a gel on which one sample is applied along the whole width of the gel) were also found useful in the screening of monoclonal antibodies. A transfer may be cut into thin (5 mm) longitudinal strips, and each strip can be used individually for antibody overlays. With narrow filter strips (that still represent the complete protein pattern of interest), the volume of reagents required are greatly reduced. In fact, by placing such a filter strip along the diagonal of a rectangular piece of nylon screening (3 X 20 cm) and by rolling up the assembly along the length of the rectangle, a well-supported filter strip of helical configuration (3 cm high) is obtained. This helical filter strip can be placed in a small scintillation vial and reacted (under rotation) with as little as 2 ml of reagent. In this manner a large number of strips can be processed simultaneously. To improve the transfer technique, we have tried to make it more sensitive and more amenable to quantitation than is possible by current procedures. This intent demanded finding conditions for efficient elution of proteins from gels and maximal retention of transferred protein on filters. The electrophoretic elution of proteins from SDS-polyacryalmide gel electrophoretograms in buffer containing methanol has been shown to be efficient (>90% recovery) only when electrophoresis is carried out for 16 to 22 h (4). We have shown that the omission of methanol from the transfer buffer increases the elution efficiency of proteins from SDS-polyacrylamide gel. However, under such conditions the protein-binding capacity of NC is greatly exceeded. Within
404
GERSHONI
15 min of electrophoretic transfer of ‘2sI-labeled BSA in the absence of methanol, protein can be detected on three sequential layers of NC. After 1 h, - 10% of the protein eluted could be found on the last of the five sequential layers of NC, and after 2 h of transfer, protein was shown to flow through 10 NC layers (Fig. 3). Under the same transfer conditions, ZB was found to be considerably more effective than NC as an immobilizing matrix: in seven experiments carried out in parallel, 72.15% f 4.73 (SE) of the protein eluted from each gel (as defined in Fig. 3) was recovered on the first ZB filter, as opposed to 20.35% f 2.24 (SE) found on the first NC filter. On account of the highbinding capacity of ZB for proteins, the rate (and extent) of protein elution from gels can be increased, the time needed for transfer can be greatly reduced, and larger loads of proteins can be handled. Moreover, as the retention of transferred proteins on ZB is superior to that on NC, higher signals are obtained rendering the overlay techniques more sensitive (Figs. 6 and 7). Due to its high affinity for anions, ZB may prove to be extremely useful for RNA and DNA gel analyses. Indeed, preliminary results show that [32P]RNA binds well to ZB. The high binding capacity of this material has, however, some disadvantages. Relatively long treatment (12 h) at high temperature (45-50°C) with either 10% BSA or 1% hemoglobin is required to insure effective quenching of unbound sites. These conditions should not be detrimental for most proteins. In fact, most often protein samples are boiled prior to electrophoresis. In addition, protein patterns on ZB filters cannot be easily stained because most dyes commonly used as protein stains are themselves anionic (e.g., Coomassie brilliant blue or amido black 10B) and bind to the filter. As yet no simple method for staining proteins on ZB has been found, although many dyes, reagents, and different approaches have been tried (see Materials and Methods).
AND
PALADE
ABCDEFG FIG. 7. ‘*sI-labeled lectin overlay of electrophoretic transfers from SDS-polyacrylamide gel electrophoretograms of murine erythrocytic ghosts. Aliquots of murine erythrocytic ghosts (25 pg each) were resolved on a 10% SDS-polyacrylamide gel and electrophoretically transferred to either NC or ZB filters for 2 h in Trisglycine buffer at 200 mA. Lane A is the original electrophoretogram stained with Coomassie brilliant blue. Lane B is a Coomassie brilliant blue stained electrophoretogram after transfer to an NC filter. Lane C is the corresponding NC transfer stained with amido black 10B. Lane D is an autoradiogram of a hemoglobinquenched NC transfer overlayed 1 h with ‘251-labeled concanavilin A (5 X 10’ cpm total) and washed as described under Materials and Methods. Lane F is the same filter after washing in succession with a-methylglucoside (100 mM in PBS) and cy-methylmannoside (100 mM in PBS). Lanes E and G are ZB transfers treated in the same way as lanes D and F, respectively.
ELECTROPHORETIC
TRANSFER
In conclusion, ZB appears to be more useful than other available filter materials for SDS-polyacrylamide gel electrophoretogram transfers and subsequent overlay techniques, because of its high and tenacious binding capacity for proteins. Binding properties of this type are of critical importance in transfer technology.
ACKNOWLEDGMENTS We thank Dr. Pietro DeCamilli, Ms. Penny Miller, and Dr. Carl Roman0 for providing samples of bovine brain homogenates and anti-protein I; Dr. Ira Mellman for useful advice “with protein radioiodination; Mrs. Cindy Davis for typing the manuscript; and Dr. Zaid Smith for sustained critical discussion. This work was supported by NIH Grant GM 27303 and by a Chaim Weizmann Postdoctoral Fellowship (J. M. G).
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Glass, W. F., Briggs, R. C., and Hnilica, L. S. (1981) Science 211, 70-72.
OF PROTEINS
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6. Glass, W. F., Briggs, R. C., and Hnilica, L. S. (1981) Anal. Biochem. 115, 219-224. 7. Renart, J., Reiser, J., and Stark, G. R. (1979) Proc. Nat. Acad. Sci. USA 76, 3116-3120. 8. Stellwag, E. J., and Dahlberg, A. E. (1980) Nucl. Acids Res. 8, 299-3 17. 9. Symington, J., Green, M., and Bra&man, K. (1981) Proc. Nat. Acad. Sci. USA 78, 177-181. 10. Burridge, K. (1978) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 50, pp. 54-64, Academic Press, New York. 11. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617. 12. DeCamilli, P., Ueda, T., Bloom, F. E., Battenerg, E., and Greengard, P. (1979) Proc. Nat. Acad. Sci. USA 76, 5977-598 1. 13. Fraker, P. J., and Speck, J. C. (1978) Biochem. Biophys. Rex Commun. 80, 849-857. 14. Laemmli, I-l. K. (1970) Nature flondon) 227,680685.
15. Sammons, D. W., Adams, L. D., and Nishizawa, E. E. (1981) Electrophoresis 2, 135-141. 16. Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) Anal. Biochem. 98, 231-237. 17. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Eiochem. 105, 361-363. 18. Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1980) Science 211, 1437-1438. 19. Troyer, H. (1980) in Principles and Techniques of Histochemistry, 1st ed., Little, Brown, Boston. 20. Wallis, C., Melnick, J. L., and Gerba, C. P. (1979) Annu. Rev. Microbial. 33,413-437. 21. Schneider, Z. (1980) Anal. Biochem. 108,96-103. 22. Farrah, S. R., Shah, D. O., and Ingram, L. 0. (1981) Proc. Nat. Acad. Sci. USA 78, 12291232.