Detection of Proteins after Biotinylation within Polyacrylamide Gels (BIG Method)

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8. Schroeder, W. T., Thacher, S. M., Stewart-Galetka, S., Annarella, M., Chema, D., Siciliano, M. J., Davies, P. J. A., Tang, H.-Y., Sowa, B. A., and Duvic, M. (1992) Type I keratinocyte transglutaminase: Expression in human skin and psoriasis. J. Invest. Dermatol. 99, 27–34. 9. Barnes, A. R., and Sugden, J. K. (1990) Comparison of colourimetric methods for ammonia determination. Pharm. Acta Helv. 65, 258 –261. 10. Sheng, S., Kraft, J. J., and Shuster, S. M. (1993) A specific quantitative colorimetric assay for L-asparagine. Anal. Biochem. 211 242–249. 11. Kun, E., and Kearney, E. B. (1974) in Methods of Enzymatic Analysis (Lund, P., Ed.), pp. 1802–1806, Academic Press, New York. 12. Folk, J. E., and Chung, S. I. (1985) Transglutaminases. [review] Methods Enzymol. 113, 358 –364. 13. Lowry, D. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. 14. Achyuthan, K. E. (1998) Enzymatic and kinetic properties of blood coagulation factor XIIIa and guinea pig liver Transglutaminase utilizing (6-[N-(4-aminobutyl)-N-ethylamino]-2,3-dihydrophthalazine-1,4-dione, as a novel, specific and sensitive chemiluminescent substrate. J. Biolumin. Chemilumin. 13, 1–11. 15. Gorman, J. J., and Folk, J. E. (1980) Structural features of glutamine substrates for human plasma factor XIIIa (activated blood coagulation factor XIII) J. Biol. Chem. 255 419 – 427. 16. Fernandez-Patron, C., Castellanos-Serra, L., and Rodriguez, P. (1992) Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: Sensitive detection of unmodified proteins. BioTechniques 12, 564 –573. 17. Gross, M., Whetzel, N. K., and Folk, J. E. (1975) The extended active site of guinea pig liver transglutaminase J. Biol. Chem. 250, 4648 – 4655. 18. Olson, J. A., and Anfinsen, C. B. (1953) Kinetic and equilibrium studies and crystalline L-glutamic acid dehydrogenase J. Biol. Chem. 202, 841– 856. 19. Gore, M. G., Greenwood, C., and Holbrook, J. J. (1972) Studies on the glutamate dehydrogenase from Neurospora crassa Biochem. J. 127, 30p–31p.

Detection of Proteins after Biotinylation within Polyacrylamide Gels (BIG Method) 1 Wolfgang W. A. Schamel 2 Department of Molecular Immunology, Biologie III, University of Freiburg and Max-Planck-Institut fu¨ r Immunbiologie, Stu¨ beweg 51, 79108 Freiburg, Germany Received May 10, 1999

The most commonly used protocol to analyze proteins is their separation by sodium dodecyl sulfate– 1

This work was supported by the Deutsche Forschungsgemeinschaft through SFB 388 and the Leibniz Prize to M. Reth. The BIG Analytical Biochemistry 274, 144 –146 (1999) Article ID abio.1999.4238 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

polyacrylamide gel electrophoresis (SDS–PAGE) and subsequent staining of the sample. Silver staining is regarded as the most sensitive method, allowing the detection of 0.1 to 10 ng of a protein (1– 4). Previously our lab has developed a method for the biotinylation of proteins on the sorbent (5). Here I describe a biotinbased procedure that stains proteins in gels (BIG 3 method) with a detection sensitivity of 40 pg. Materials and Methods Biotinylation in solution (Fig. 1a). Bovine serum albumin (BSA) was incubated for 15 min with 1 mg/ml NHS– biotin (EZ-Link sulfo-NHS– biotin, Pierce) in phosphate-buffered saline (PBS). After quenching for 15 min with 100 mM Tris–HCl, pH 7.2, biotinylated BSA was size-separated on a 10% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes as described before (6) at 200 V for 15 min. After blocking with 5% skimmed milk in PBS/Tween (PBS, 0.1% Tween 20), membranes were incubated with horseradish peroxidase-conjugated streptavidin (streptavidin–HRPO, Amersham) 1:2000 in PBS/ Tween for 30 min and washed as depicted in Table 1. The ECL system (Amersham) was used for detection. Biotinylation within the gel (Fig. 1b). BSA was separated as before. Biotinylation was done as shown in Table 1 (steps 1–5), except for step 4: 1 mg/ml NHS– biotin for 1 h. Biotinylated BSA was blotted and detected as described above. Biotinylation on the membrane (Fig. 1c). BSA was separated and blotted as before. The membrane was washed with PBS, incubated for 1 h with 4 ml of 1 mg/ml NHS– biotin in PBS, washed with PBS, incubated 30 min with 100 mM Tris–HCl, pH 7.2, and washed. Blocking of the membrane and development were done as described above. Silver stain (Fig. 1d). The gel was developed using the silver stain plus kit (Bio-Rad) according to the instructions and exposed to a duplicating/RA film (Kodak). The BIG method (Fig. 1e). The BIG method is described in Table 1. Rotiblock was purchased from Roth and the ABC system (ImmunoPure ultrasensitive ABC staining kit) was from Pierce.

protocol received German Patent No. 19913940.7 from the German Patent and Trademark Office on March 26, 1999. 2 To whom correspondence should be addressed. Fax: 49-761-5108423. E-mail: [email protected]. 3 Abbreviations used: ABC, avidin– biotin complex; BIG, Biotinylation In the Gel; BSA, bovine serum albumin; HRPO, horseradish peroxidase; DTT, dithiothreitol; NHS– biotin, sulfosuccinimido– biotin; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride.

145

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Results and Discussion BSA was biotinylated at different steps of the Western blotting protocol. The resulting sensitivities were compared with silver staining. The sample was biotinylated before (Fig. 1a) or directly after (data not shown) its denaturation. After separation, blotting, and development, both procedures resulted in a detection limit of 5 ng of BSA. If BSA was biotinylated in the gel before transfer to a membrane, 1 ng was detected (Fig. 1b). Biotinylation on the membrane (Fig. 1c) resulted in a sensitivity (10 ng), which was similar to that of two silver stain procedures [Fig. 1d and data not shown (1)]. Biotinylation of proteins on the membrane and probing with streptavidin have been published (7). In accordance with my result (Fig. 1c), a sensitivity similar to silver staining was achieved. The sensitivity of biotinylation within the gel was superior; therefore, this protocol was optimized (data not shown). To reduce samples, dithiothreitol (DTT, 10 mg/ml) should be used and quenched with iodoacetamide (50 mg/ml, both for 10 min at room temperature) before loading of the samples, in order to avoid background signals of around 60 kDa. The gels must be dialyzed to remove glycine and Tris, otherwise the reaction with NHS– biotin is quenched. Dialyzing times between 0.5 and 3 h were tested, with the strongest signal at 3 h (1 mg/ml NHS– biotin). The NHS– biotin concentration was varied between 0.01 and 3 mg/ml with an optimum at 0.75 mg/ml. The longer this incubation, the better the signal with a plateau at 2 h. Quenching or removal

FIG. 1. Sensitivities of protein detection by the different biotinylation-based techniques and silver staining. Twofold dilutions of BSA were used for each SDS–PAGE as indicated at the top. BSA was biotinylated before loading the gel (a), after its separation within the gel (b), or after its transfer to the membrane (c). After blocking, membranes were reacted with streptavidin–HRPO and developed with the ECL system. A silver stain kit was used in d. In e, SDS–PAGE-separated BSA was detected using the BIG method (Table 1).

TABLE 1

Protocol for the BIG (Biotinylation In the Gel) Method Step

Gel/membrane treatment a

0

Quench DTT with iodoacetamide before running of the gel Dialyze gel (80 ⫻ 40 ⫻ 1 mm) in 50 ml PBS Rinse gel once with H 2O Repeat steps 1 and 2 five times Incubate gel in 5 ml of 0.75 mg/ml NHS–biotin in PBS Transfer proteins to nitrocellulose Block membrane with 1⫻ Rotiblock Incubate membrane with the ABC system 1:10 in PBS/Tween Wash membrane in 10 ml PBS/Tween Rinse membrane once with PBS/Tween Repeat steps 8 and 9 two times Develop membrane with ECL

1 2 3 4 5 6 7 8 9 10 11

Time (min)

30 150 120 15 50 30 5 10 Sum: 7 h

a

All incubations were done at room temperature (except step 5) and with constant agitation (except steps 5 and 11).

of the unreacted NHS– biotin was not necessary before the transfer step because biotin itself did not bind to membranes (data not shown). Comparing PVDF and nitrocellulose membranes showed a higher sensitivity with nitrocellulose. Blocking of the membrane was tested with 5% skimmed milk, 2% BSA, 0.5% gelatin, or Rotiblock in PBS/Tween, the latter being the best. For probing of the membranes, different concentrations of the ABC system or streptavidin–HRPO were tested. The ABC kit is a mixture of the biotinylated HRPO enzyme and the tetravalent avidin, generating large complexes that can be bound to one single biotin molecule. Therefore, the number of enzyme molecules per biotinylated protein is increased in comparison to streptavidin–HRPO, leading to higher sensitivity (data not shown). Here I report the application of the ABC system for Western blotting at a dilution of 1:10. These optimized conditions are summarized in Table 1. The technique which I term the BIG method (Biotinylation In the Gel) could visualize 40 pg of BSA (Fig. 1e) and was applicable to all proteins analyzed so far. The BIG method was used for native gels (data not shown) and does not lead to a molecular weight shift of proteins, as seen if the proteins were biotinylated before their separation (8). The identical membrane can be used for subsequent immunostaining using specific antisera (data not shown). The sensitivity of the BIG method may further be enhanced by utilization of NHS–(biotin) 6 (9) or of a photoactivatable biotin in order to enhance the degree of biotinylation of the proteins. Here I report a protein stain based on the high affinity between biotinylated proteins and streptavidin.

146

NOTES & TIPS

This protocol, the BIG method, detects proteins with a 100 times higher sensitivity than commonly used silver stains. Acknowledgments. I thank Bernd Wollscheid for his technical comments and Karin R. Schamel, Drs. Robert Moore, Pete J. Nielsen, and Michael Reth for advice and reading the manuscript.

REFERENCES 1. Heukeshoven, J., and Dernick, R. (1988) Electrophoresis 9, 28 – 32. 2. Merril, C. R., Dunau, M. L., and Goldman, D. (1981) Anal. Biochem. 110, 201–207. 3. Moeremans, M., Daneels, G., and De Mey, J. (1985) Anal. Biochem. 145, 315–321. 4. Ross, M., and Peters, L. (1990) Biotechniques 9, 532–533. 5. Kim, K. M., Adachi, T., Nielsen, P. J., Terashima, M., Lamers, M. C., Ko¨ hler, G., and Reth, M. (1994) EMBO J. 13, 3793–3800. 6. Adachi, T., Schamel, W. A., Kim, K. M., Watanabe, T., Becker, B., Nielsen, P. J., and Reth, M. (1996) EMBO J. 15, 1534 –1541. 7. LaRochelle, W. J., and Froehner, S. C. (1986) J. Immunol. Methods 92, 65–71. 8. Lynch, G. W., Dearden, M., Sloane, A. J., Humphery-Smith, I., and Cunningham, A. L. (1996) Electrophoresis 17, 227–234. 9. Hofmann, K., Titus, G., Montibeller, J. A., and Finn, F. M. (1982) Biochemistry 21, 978 –984.

Charge Heterogeneity of Commercial, Red-Shifted Recombinant Green Fluorescent Protein, Revealed by Capillary Zone Electrophoresis under Nondenaturing Conditions Sergey P. Radko,* ,1 Miroslava Stastna,* Zsuzsanna Buzas,* ,2 David Kingsley,† and Andreas Chrambach* ,3 *Section on Macromolecular Analysis and †Section on Membrane and Cellular Biophysics, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1580 Received June 10, 1999

The conventional electrophoretic method of protein analysis is SDS–PAGE. In its ubiquitous applications, the fact is frequently neglected that in the 1 Permanent address: Research Center for Medical Genetics, Russian Academy of Medical Science, Moscow, Russia. 2 Permanent address: Dept. of Biochemistry, Agricultural Biotechnology Center, Godollo, Hungary. 3 To whom correspondence should be addressed. Fax: (301) 4020263. E-mail: [email protected].

Analytical Biochemistry 274, 146 –148 (1999) Article ID abio.1999.4240 0003-2697/99

presence of SDS, deaggregation of size isomeric forms and suppression of charge isomeric forms of the protein take place. With regard to recombinant proteins, the consequence is that biologically relevant posttranslational modifications of the gene product and(or) its modifications due to purification procedures may remain undetected. A case in point is commercial recombinant red-shifted green fluorescent protein (rEGFP) 4 exhibiting a single band in SDS–PAGE. This protein is used to tag proteins and biologically relevant particles to monitor their transport in living cells and(or) to detect them during isolation and purification (e.g., 1 and references therein). Materials and methods. Recombinant GFP (rGFP) was prepared from transiently transfected Sf9 cells (American Type Tissue Culture Collection, Manassas, VA). rGFP was constitutively expressed under the control of an optimized OpMNPVgp64 early promoter (p64CAT-166) (2) in SF9 cells using the pGFP-Bac plasmid. pGFP-Bac was constructed by modification of the pATH-1 expression plasmid. Briefly, pATH was digested with HpaI and HindIII, the large fragment was ligated to a linker containing NcoI and SmaI sites. Following digestion with NcoI and SmaI, the NcoI–StuI fragment encoding red-shifted GFP from plasmid pEGFP (Clontech Inc., Cat. No. 6077-1) was ligated to create pGFP-Bac. Cells were harvested 24 to 48 h posttransfection and cytosol proteins including rGFP were released into solution by osmotic shock (by pelleting and consequently resuspending the cells in distilled water). Cell debris was pelleted at 2000g for 30 min and the buffer concentrate was added to the collected supernatant to adjust it to 50 mM Tris, 25 mM CHES, pH 9.0 (TC buffer). Thereafter, the solution was centrifuged at 10,000g for 15 min, supernatant was collected, diluted 10-fold with TC buffer and used as a sample for capillary zone electrophoresis (CZE). Commercial red-shifted recombinant green fluorescent protein, rEGFP (as designated by the manufacturer), was obtained from Clontech Labs. (Palo Alto, CA, Cat. No. 8365-1, Lot No. 9010585) as a 1.0 mg/ml solution in 10 mM Tris–HCl (pH 8.0), 10 mM EDTA. Upon 100-fold dilution with TC buffer, the solution was used as a sample for CZE. CZE was carried out in a P/ACE-2100 apparatus (Beckman Instruments, Fullerton, CA) equipped with fluorescence detector. Excitation/emission wavelengths were 488/520 nm. Solutions of linear polyacrylamide of molecular weight of 350,000 (Polysciences, Inc., Warrington, PA, Cat No. 19790, designated as PA-350) were prepared in TC buffer and applied to CZE as described earlier (3). Fused silica capillaries 4

Abbreviations used: rEGFP, red-shifted green fluorescent protein; rGFP, recombinant GFP; CZE, capillary zone electrophoresis.