Photobiotin as a sensitive probe for protein labelling

ANALYTICAL

BIOCHEMISTRY

163, 15 l- I58 (1987)

Photobiotin

as a Sensitive

Probe for Protein Labeling

ERNEST LACEY’ AND WARWICK N. GRANT CSIRO, Division of Animal Health, M&aster

Laboratory, Private Bag No. 1, P.O. Glebe NSW 2037, Australia

Received October 6, 1986 A sensitive method for the nonisotopic in vitro labeling of proteins under physiological conditions using photobiotin, a compound originally developed for labeling nucleic acids (Forster et al. (1985) Nucleic Acids Rex 13,745) has been developed. Using sheep brain tubulin as a model protein it was shown that labeling with photobiotin resulted in detection limits below 10 pg when avidin-alkaline phosphatase was used in the final step. No significant loss of tubulin polymerization, colchicine binding, recognition by antitubulin antibodies, or changes in electrophoretic behavior were observed. In addition, photobiotinylation of antitubulin antibodies did not affect their recognition of unlabeled tubulin. The use of photobiotin labeling with avidin-alkaline phosphatase detection for electrophoretic separations of molecular weight standards, crude protein supematants, and tubulin gave a 64 to 1024-fold increase in sensitivity over Coomassie blue staining. 0 1987 Academic press, Inc. KEY WORDS: photobiotin; protein labeling; tubulin; dot blot; photoactivation; antibody-antigen interactions.

Methods for labeling proteins are an important aspect of many facets of biochemistry, pharmacology, and immunology. Such techniques have principally involved the incorporation of radioactive isotopes (1). However, the inherent dangers in the use of the high specific activities required together with problems of self-radiolysis, isotope stability, and protein denaturation during labeling have led to the development of alternative labeling procedures. Based on the versatility and sensitivity of biotin-avidin (or streptavidin) linked enzyme techniques, a series of derivatized biotin reagents possessing highly reactive electrophilic sites has been developed (2). These reagents possess clear advantages in terms of safety, stability, and ease of handling over radionuclide incorporation. However, covalent interaction with proteins occurs at specific sites, notably -NH2, -OH, and -SH residues of polar amino acids. While the role of these sites in the relevant fields of investigation is not always known, such residues are ’ To whom correspondence should be addressed.

generally considered to play crucial roles in the “native” conformation of proteins, particularly for drug-protein, substrate-protein, and protein-protein interactions (3,4). Consequently these procedures may adversely affect the biological functions of labeled proteins. Photoactive reagents, particularly aromatic azides, react covalently via nitrene intermediates with low specificity for particular amino acids thereby minimizing the chances of reaction at a specific site (5). Recently, photobiotin (N-(4-azido-2-nitrophenyl)-N’-(IV-biotinyl-3-aminopropyl)-N’methyl- 1,3-propanediamine) acetate, a water soluble, photoactive linked biotin reagent, was developed for DNA and RNA labeling as an alternative to existing 32P labeling techniques (6). The nonspecific nature of photoactivation as a labeling technique and the high sensitivity (comparable to 32P) (6) achieved are ideal prerequisites for a generally applicable protein labeling reagent. To examine this proposition, we investigate the conditions for optimal labeling of proteins with photobiotin (photobiotinyla151

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Copyright 0 1987 by Academic F’ress, Inc. All rights of reproduction in any form reserved.

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LACEY AND GRANT

tion) using the ubiquitous eukaryotic structural protein, tubulin. Particular issues addressed are the sensitivity of detection and the maintenance of biological, pharmacological, and immunological activity. The use of photobiotin as a general label for the detection of proteins after electrophoresis is compared with Coomassie blue staining.

philized mixture with sucrose (27 mg) from Pharmacia, Australia. Isolation of tubulin. Crude supematants of sheep brain were prepared in 0.025 M MES buffer containing 1 mM EGTA, 1 mM GTP, and 0.5 mM magnesium sulfate as described previously (7). For polymerization studies, tubulin was isolated by temperature-dependent polymerization-depolymerization from MATERIALS AND METHODS the crude supematant (7). Preparation of dimerit tubulin (purity > 95%) for dot blot and Materials. Photobiotin was either purelectrophoretic studies was achieved using an chased from BRESA (G.P.O. Box 498, Adearginine linked Sepharose 4B column (Pharlaide, South Australia, 5001) as the acetate macia), eluted as previously described (8). salt or synthesized according to the method Photobiotinylation. A solution of the ap of Forster et al. (6). Synthetically prepared propriate protein(s) in either PBS or MES photobiotin was prepared in aqueous solubuffer in a plastic Eppendorf tube was vortion by dissolution in a molar equivalent of texed with photobiotin and placed on ice; the acetic acid and gradual dilution with distilled opened tube was irradiated (National Selfwater to the required concentration. Ballasted BHRF 240-250 V 250 W-P lamp) Avidin-alkaline phosphatase conjugate at a distance of 20 cm in a vertical position (avidin-AP): BCIP, ,NBT, MES, EGTA, and for 20 min. Norit A animal charcoal were obtained from Examination of the effect of time of irraSigma. Streptavidin-HRPO, LY-,and Btubudiation, volume of solution, protein concenlin monoclonal antibodies (supplied as tration, and molarity of photobiotin was unascites fluid) and t3H]CLC were purchased dertaken by appropriate modification of this from Amersham, Australia. Nitrocellulose procedure. (0.45 pm pore size), Coomassie blue dye Polymerization of tub&n. A tubulin soluconcentrate, and all electrophoretic buffers tion reconstituted from microtubules (stored and reagents were obtained from Bio-Rad, in liquid nitrogen (7)) was mixed with phoAustralia. GTP was purchased from tobiotin (0 to 100 Meqs), diluted to 1 mg/ml Boehringer-Mannheim, Australia. All other with MES buffer, and irradiated. Immedireagents were obtained from Ajax Chemiately after photobiotinylation, GTP was cals, Australia, and were of analytical reagent added and the polymerization of tubulin was grade. monitored spectrophotometrically (Unikon Molecular mass standards, phosphorylase 820 Spectrophotometer, Kontron, SwitzerB (94 kDa, 64 pg), BSA (67 kDa, 84 lg), land). Polymerization was initiated by ovalbumin (43 kDa, 147 pg), carbonic anhy- warming to 37°C and the absorbance change drase (30 kDa, 83 pg), soybean trypsin inhibwas monitored until control samples reached itor (20.1 kDa, 80 I.cg), and a-lactalbumin equilibrium turbidity (16 min). (14.4 kDa, 12 1 pg), were obtained as a lyo[3H]Colchicine binding. Photobiotinylated tubulin solutions ( 100 ~1 volume) were incubated with [‘H]CLC (10 PM, sp. act. = 0.19 2 Abbreviations used: AP, alkaline phosphatase; for 1 h at 37°C in a shaking water BCIP, 5-bromo-4chloro-3-indolyl phosphate; BSA, bo- Ci/mmol) vine serum albumin; CLC, colchicine; DAB, 3,3’-diamibath. The binding was terminated by a 5-min nobenzidine; GTP, guanosine triphosphate; HRPO, incubation with 0.5 ml of a 2 m&nl suspcnhorse radish peroxidase; Mab, monoclonal antibody; sion of charcoal in 1% BSA. After charcoal Meq, molar equivalent; NBT, nitroblue tetrazolium; (centrifugation, 10,000 i-pm for PBS-T, 0.05% Tween 20 in phosphate-buffered saline; precipitation 10 min), the bound radioactivity was deterSDS, sodium dodecyl sulfate.

PHOTOBIOTIN

PROTEIN

mined by liquid scintillation spectroscopy of a supematant sample (9). Calculation of the percentage of inhibition of [3H]CLC binding was obtained as a ratio of photobiotinylated tubulin binding to control binding. Dot blot. Examination of the detection limits of photobiotinylation was undertaken by serial (1:2) dilution of the sample over the range 100,000 to 0.4 rig/ml (generally 14 to 18 dilutions were used). An amount of each dilution (2.5 ~1) was applied to a ruled matrix (1 X 1 cm’) of nitrocellulose filter and allowed to dry; the filter was blocked with 5% BSA in PBS-T for 10 min on a rotatory shaker. The filter was lightly washed with PBS-T to remove excess BSA, mounted on a hydrophobic film (Parafilm, HIS Scientific, Australia) supported on a glass plate in a moist chamber, and incubated with a 1: 1000 dilution of avidin-AP (ca. 1 pg/ml in PBS-T) or streptavidin-HRPO for 30 min. Unbound reagent was removed by 3 X 100 ml PBS-T washes for 10 min each. For the avidin-AP incubations a fourth wash with 0.1 M Tris containing 100 mM sodium chloride and 5 IrIM magnesium chloride at pH 9.5 was used. Streptavidin-HRPO color development was carried out with DAB or DAB with cobalt chloride and nickel ammonium sulphate ( 10) while avidin-AP color development using BCIP/NTB was carried out according to Forster et al. (6) on parafilm in the dark for 1 h. Antibody-antigen interactions. Unlabeled (Y-and /3-tubulin Mabs, serially (1:2) diluted in the range of 1:250 to 1:8000 were prepared and 2.5 ~1 samples were applied to a ruled matrix (1 X 1 cm2) on nitrocellulose filter. The filter was blocked with 5% BSA in PBS ( 10 min), washed with PBS, and incubated with photobiotinylated tubulin (0, 0.1, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, and 100 Meqs, 2.5 &ml) for 1 h on paratilm (11). The filter was then washed with 4 X 100 ml PBS and reacted with avidin-AP in PBS. All subsequent procedures were as described for dot blot except that only PBS (not PBS-T) was used.

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The reverse procedure was undertaken by application of serial (1:2) dilutions of 2.5 ~1 of unlabeled tubulin ( 125 to 1.9 ng) to nitrocellulose and by subsequent incubation with photobiotinylated (Y- and 8-tubulin Mabs. Labeled Mabs were prepared by irradiation of a 1:2500 dilution (100 ~1) with 20 Meqs photobiotin for 20 min. A 1: 16 dilution of labeled Mab (i.e., total dilution 1:40,000) was applied to the nitrocellulose filters, incubated for 1 h, and then washed and reacted with avidin-AP as above. Electrophoresis. Solutions of photobiotinylated molecular weight standards (500 pg/ml), crude sheep brain supematant (1000 &ml), and tubulin (50 &ml) were diluted with 1.5 vol of 0.125 M Tris (pH 6.8) containing 10% glycerol, 5% 2-mercaptoethanol, 2.5% SDS, and 0.1% bromphenol blue and heated at 90°C for 2 min. The boiled solutions were serially ( 1:4) diluted to give 1:4, 1: 16, 1:64, 1:256, and 1:1024 dilutions with the Tris buffer and 25 J was applied to two 16 X 18 X 0.15~cm gels containing a 3% (w/v) acrylamide stacking gel and 10% (w/v) acrylamide resolving gel (12). The gels were electrophoresed in parallel for 5 h at 22-25 mA/gel, constant current. After this the first gel was stained with 0.25% Coomassie blue R250 in water containing 25% isopropanol and 10% glacial acetic acid for 2 h and then destained by repeated washing in water containing 7% glacial acetic acid and 5% methanol. The second gel was electrophoretically transferred to nitrocellulose ( 13) and stained with avidin-AP as described for the dot blot. Protein determination. All protein concentrations were determined using the commercially available Coomassie blue dye concentrate according to Bradford ( 14). RESULTS

Efect of photobiotinylation on tub&n polymerization. In the absence of photobiotin and irradiation the polymerization of tubulin reached equilibrium at 16 min, monitored as an increase in absorbance at 350 nm

154

LACEY AND GRANT

0

1

2

3 4

5 6

,

6 4 10 1, 12 13 v. 15 16 Time(m)

FIG. 1. Effect of variation in the molar ratio of photobiotin on the in vitro polymerization of sheep brain tubulin. Tubulin solutions (3.85 mg/mI, 260 ~1) were mixedwith O(light),O.l, 0.5, 1.0,2.0,5.0, 10,20,50, and 100 Mcq of photobiotin (3.716 nmol/pl), diluted to 1 mg/ml (10 pmol) and irradiated for 20 min. A duplicate sample without photobiotin was stored on ice in foil (dark). A sample (0.7 ml) of each tube was taken, mixed with 50 ~1 GTP (1 .O mg/ml) in 1.5 ml polystyrene cuvettes, and warmed to 37°C for 16 min. The increase in turbidity was monitored spectrophotometrically at 350 nm. Absorbance at time = 0 was normalized to zero to account for the absorbance of photobiotin at 350 nm. The percentage of inhibition of polymerization was derived from the extent of polymerization at 16 min for each ratio of Meqs of photobiotin ( 1 to 100) compared to that of the light-exposed tubulin control. The KS0 (6.9 Meqs) was derived from a plot of the percentage of inhibition versus log photobiotin Meqs and interpolation at the 50% value. (correlation coefficient, r = 0.998).

due to the turbidity of insoluble microtubules (absorbance change = 0.275 AU, Fig. 1). Exposure to light had no significant effect on either the rate (absorbance change over the linear phase) or the extent of polymerization (reproducibility of polymerization profiles is f 10% (7)). Irradiation of tubulin with 0.1, 0.5, and 1 .O Meqs photobiotin resulted in only minimal changes compared with the control profiles with less than 10% inhibition in the extent of polymerization. At 2.0 Meqs, 12.5% inhibition in the extent was observed with no significant change in the rate. However at 5.0 Meqs a marked effect on the extent (40% inhibition) and rate (55% inhibition) was observed. This inhibition increased with increasing photobiotin concentration.

Calculation of the I& value (7) for the extent of polymerization by graphical interpolation of a percentage of inhibition versus log photobiotin Meqs plot gives 50% inhibition at 6.7 Meqs. Eflect of photobiotinylation on [3H]CLC binding. Investigations of [3H]CLC binding to samples of tubulin photobiotinylated in the polymerization study are shown in Fig. 2. At concentrations less than 2 Meqs no inhibition was observed and only 7% inhibition was observed at 5 Meqs. However, further increases inhibited [3H]CLC binding in a log-linear manner, giving an interpolated 50% inhibition value at 76 Meqs of photobiotin. Minimum detection limits. A preliminary comparison of the avidin-AP and streptavidin-HRPO detection systems for photobiotinylated tubulin demonstrated that the use of the latter conjugate with DAB alone was I28-fold less sensitive than avidin-AP and 32-fold less sensitive with cobalt-nickel enhancement (10) at all levels of photobiotin labeling (data not shown). For streptavidinHRPO reactions no background binding to the unlabeled tubulin was observed at 2500 pg (data not shown), while for avidin-AP low intensities of background binding were ob-

Oi

0

-10

-0.5 0 Lcgx) Photobmtin

0.5 10 molar equrvalents

1-5

20

FIG. 2. Effect of variation of the molar ratio of photobiotin on [‘Hlcolchicine binding to sheep brain tubulin. Duplicate samples of 90 ~1 of photobiotinylated sheep brain tubulin (90 pg) from the experiment detailed in Fig. 1 were incubated with 10 ~1 r3H]CLC ( 10 PM) for 1 h. [‘H]CLC was calculated by counting the supematant fraction after charcoal extraction and precipitation. Binding was expressed as a percentage of the control (photobiotin = 0) incubation. Duplicate determinations varied by less than 5% of the quoted value.

PHOTOBIOTIN

PROTEIN

served down to 15.6 ng. However, over the range of 125 to 15.6 ng, the increase above background can clearly be identified (Fig. 3). Increasing the ratio of photobiotin to tubulin increases the sensitivity of detection. Ii the biological range (0.1 to 5 Meqs) tubulin can be detected down to 0.06 ng, while inactive tubulin (100 Meqs) can be identified down to as low as 0.008 ng (Fig. 3). Time of irradiation. The effect of increasing irradiation time on the extent of labeling with 5 Meqs photobiotin was examined. Due to the light-sensitive nature of the azide, zero time could not readily be achieved as limited exposure during application to the filter could not be eliminated. Thus at zero time a detection limit of 2 ng was observed. Upon irradiation for 1 and 2 min, labeled tubulin could be detected at 0.5 and 0.25 ng, respectively. The detection of tubulin down to 0.06

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LABELING

ng was achieved by 3 min. Over the range of 3 to 30 min the detection limit (0.06 ng) remained essentially constant with a gradual increased coloration of spots above this limit ‘q. 13 to 1 ng) for lo- to 30-min incubations ..rth respect to the 6-min irradiation (data not shown). Volume and protein concentration of labeled tub&n. The labeling of tubulin solutions at a constant protein concentration (100 &ml) in volumes from 30 to 3000 ~1 with 5 Meqs photobiotin gave detection minima at 0.06 ng of applied protein. Variation of the protein concentration over the range of 1 to 2000 pg in 500 ~1 gave identical detection limits. Eflect on antigenicity. When dilutions of both CX-and P-tubulin unlabeled Mabs are bound to nitrocellulose and incubated with labeled tubulin according to the method of

0

125 62.531..315.6

7.8 3.9 Quantity

2.0 1.0

0.5 0.25 0.u O-060.03 0~1.5~08

of Tubulin *lied

(ng)

FIG. 3. Effect of the molar ratio of photobiotin to tubulin on the minimum detection level observed with the dot blot technique. Samples (2.5 ~1) of serial 1:2 dilutions of each labeling reaction (0.1-100 Meqs) were applied to a nitrocellulose filter and then blocked with BSA and incubated with avidin-AP. Following washing to remove unbound avidin-AP the filter was incubated with BCIP-NBT substrate for 1 h in the dark.

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LACEY AND GRANT

125

62.5

31.25 Quantity

15.6 of Tubulin

7.8 Applied

3.9

1.9

(ns)

FIG. 4. Interaction of photobiotinylated LY-and j3monoclonal antibodies with tubulin using the da& blot procedure. Samples (2.5 ~1) of unlabeled tubulin (125-1.9 ng) were applied to nitrocellulose filters and reacted with labeled Mabs ( 1:40,000 dilution), followed by avidin-AP detection.

Suresh and Milstein (11) the formation of the antibody-antigen complexes can be readily identified. However, some limitations to this technique were observed with nonspecific background increasing with the level of labeling up to 5 Meqs. Within this range LY-and P-Mabs could be clearly identified only at 1:250. Increasing the extent of labeling reduced the problems associated with the background with a commensurate increase in the detection limits for the interaction from 1:500 at 5 Meq to 1:2000 for 10 to 100 Meqs. Attempts to eliminate the background binding by modification of the technique using PBS-T or PBS containing 0.5 M sodium chloride instead of PBS completely abolished the antibody-antigen interaction. The detection of unlabeled tubulin with photobiotinylated LY-and ,&tubulin antibodies gave no significant background problems (Fig. 4). Tubulin was detected down to 3.9 ng after incubation with either cy- or ,&labeled Mabs, indicating no significant loss of antibody activity due to photobiotinylation. Electrophoretic detection. A comparative study of the relative sensitivities of Coomassie blue staining and photobiotinylation followed by avidin-AP detection is shown in Fig. 5. At 5, 10, and 0.5 pg of protein standards, crude supernatant, and tubulin, respectively, Coomassie blue gave a typical strong coloration of all proteins with the exception of tubulin. Results for the avidin-AP detection at these protein concentrations are not shown as the lanes were completely

overstained after 1 h. Even at a I:4 dilution overstaining of the crude supematant at 1 h was apparent, while for the Coomassie blue stained gel a I:4 dilution resulted in a complete loss of tubulin detection as well as a reduction in staining intensity of the lowermolecular-weight crude supernatant proteins. No proteins were detected by Coomassie blue staining at 1: 16 while complete detection of proteins at both 1: 16 and 1:64 dilutions was observed with photobiotin:avidin-AP. Labeling enhanced the detection of tubulin in both crude and pure samples by approximately I OOO-fold over Coomassie blue, with similar increases in the detection of BSA and phosphorylase B. DISCUSSION

The use of photobiotin for the labeling of proteins is a rapid, convenient, and easily controlled procedure. For the model protein used in this study, tubulin, the extent of labeling chosen is dictated by the intended use for the labeled tubulin. Where copolymer&&on studies of labeled tubulin with unlabeled tubulin are undertaken for identification purposes (for example ( 15)) only low molar ratios of photobiotin (0.1 to 2 Meqs) are permissible. However, for pharmacological drug binding studies labeling with much higher concentrations can be achieved. These differences presumably relate to the degree of label-induced denatumtion tolerated at the specific site(s) required for polymerization and drug binding. This tolerance is dependent on the specific protein labeled and the range of biological activities required intact. Investigation of the volume and protein concentration required for labeling revealed the absence of any clear limitations. These observations are not altogether unexpected since photobiotin possesses a highly hydrophobic (water repellent) aromatic-aliphatic side chain which would tend to associate with more nonpolar environments (that is, the protein) in preference to the aqueous solution. This independence of labeling on

PHOTOBIOTIN

PROTEIN

antigen interaction occurred at high molar ratios of photobiotin (50-100 Meqs) in which tubulin is known to be devoid of biological activity. The reasons for this phenomenon are obscure since ready identification of labeled antibody-antigen interactions was observed. This suggests that the detection of unlabeled antibody by lightly labeled tubulin has been “masked” by high background binding of labeled tubulin, indicating that despite blocking with BSA, sufficient binding sites exist on nitrocellulose for the labeled tubulin to interact nonspecifically.

sample volume and concentration greatly increases the versatility of the technique. For example, dilute (ca. 2 &ml) protein solutions in 2-3 ml volumes have been successfully labeled (data not shown). Photobiotinylation of either a protein or an antibody can enable the ready identification of antibody-antigen interactions on nitrocellulose with a sensitivity compatible to the quantities of monoclonal antibody produced in culture supernatants from myeloma-spleen cell fusions. In the present study optimal detection of antibody-labeled

A.

Coomassie 1 N

B.

c

blue 1:4

T

Photobiotin

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MCT

- Avidin/

1:16 MC

1:64 T

M

C

1:254 T

M

C

1:1024 T

M

C

T

**a i

A. P.

94 67 47

FIG. 5. Comparative sensitivities of Coomassie blue staining and photobiotinylation on the detection of electrophoretic separations of various dilutions of molecular weight standards (M), crude sheep brain supernatant (C), and purified sheep brain tubulin (T).

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As a detection system for electrophoretic separations prelabeling protein solutions with photobiotin increases the sensitivity of detection by 64-fold for all proteins and for some proteins, such as tubulin, BSA, and phosphorylase B, the increase can be on the order of lOO- 1000X that of Coomassie blue. This sensitivity can be considerably increased by extending the incubation times with substrate (up to overnight without significant increases in background, data not shown). No change in electrophoretic mobility or loss of resolution was apparent following photobiotinylation. Photobiotin is superior to the Whydroxysuccinimide ester of biotin for this purpose due to its ease of use and much lower protein to protein variability in labeling efficiency (16). A comparison between avidin-AP detection of photobiotin-labeled proteins and silver staining (17) following electrophoresis was also made (Lacey and Grant, unpublished data). In our hands, tubulin stained very poorly with silver (less sensitive than Coomassie blue) and other proteins showed highly variable staining. This variability was not seen with avidin-AP detection of photobiotin-labeled proteins. For those proteins that stained well using all three methods, avidin-AP detection was approximately 4to 16-fold more sensitive than silver. Furthermore, immunoblots of photobiotinylated crude antigen mixtures can be sequentially reacted with antibody and avidin enzyme conjugates (using different enzymes or substrates for each) to allow detection of specific antigens and total protein in the same sample (data not shown). This greatly facilitates the identification of specific antigens, particularly on two-dimensional gels.

The ease of use, reliability, and versatility of photobiotin protein labeling should ensure its widespread use. ACKNOWLEDGMENTS We would like to acknowledge the assistance of Ms. Karon L. Snowdon. This work was partly funded by a grant (L/9/92 1) from the Wool Research Trust Fund on the recommendation of the Australian Wool Corp.

REFERENCES 1. The Radiochemical Centre, Amersham, England ( 1979) Technical Bulletin 79/6, l- 15. 2. Bayer, E. A., and Wilchek, M. (1980) Methods Bi@ them. Anal. 26, I-45. 3. Fraenkel-Conrat, H. (1959) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., Eds.), 2nd ed., pp. 589-6 18, Academic Press, New York. 4. Van Regenmortel, M. H. V. (1986) Trends Biothem. Sci. 11,36-39. 5. Guillory, R. J., and Jeng, S. J. (1983) Fed. Proc. 42, 2826-2830. 6.

Forster, A. C., McInnis, J. L., Skingle, D. C., and Symons, R. H. (1985) Nucleic Acids Res. 13,

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Lacey, E., and Watson, T. R. (1985) Biochem. Pharmacol. 34, 1073-1077. Iacey, E., and Prichard, R. K. (1986) Mol. Biochem. Parasitol. 19, 171-181. Lacey, E., Edgar, J. A., and Culvenor, C. C. J. Biothem. Pharmacol., in press. De Bias, A. L., and Cherwinski, H. M. (1983) Anal. Biochem. 133,214-219. Suresh, M. R., and Milstein, C. (1985) Anal. Biothem. 151, 192-195. Laemmli, U. K. (1970) Nature (London) 227,

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13. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76,4350-4354. 14. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.

15. Slabas, A. R., MacDonald, G., and Lloyd, C. W. (1980) FEBS Lett. 110, 77-79. 16. Della-Penna, D., Christoffersen, R. E., and Bennett, A. B. (1986) Anal. B&hem. 152,329-332. 17. Morrissev. J. (1981) Anal. B&hem. 117.307-310.