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NOTES & TIPS Staining and Quantification of Proteins Transferred to Polyvinylidene Fluoride Membranes Michael K. Moore and Susan M. Viselli 1 Department of Biochemistry, Midwestern University, Downers Grove, Illinois 60515 Received May 7, 1999
The ability to verify equal sample loading and uniform blotting efficiency is of major importance to quantitative immunoblotting where regulated protein expression is being evaluated. The use of an internal protein control and separate antibody for detection is not always practical and can also be expensive and time consuming. A simple, yet elegant technique employing staining with Ponceau S was described for visualization and subsequent spectrophotometric quantitation of proteins transferred to nitrocellulose (1). This method appears to be most successful when the immunodetected protein represents a small proportion of the total protein per lane (2). PVDF 2 is now often used as an alternative to nitrocellulose in immunoblotting because of its greater mechanical strength and higher protein binding capacity. The original report of the Ponceau S technique (1) involved only the use of nitrocellulose. However, a statement in Current Protocols in Immunology (3) mentions that both nitrocellulose and PVDF membranes can be reversibly stained with Ponceau S. No specifics about characteristics of PVDF membranes that allow it to be stained with Ponceau S and destained with water were provided (3). In our experience we found that the standard procedure that was developed for use with nitrocellulose was unsatisfactory with PVDF. However, with a modification of the originally described procedure we obtained reliable, quantitative results within 10 min. The original protocol involves incubating the nitrocellulose membrane containing transferred proteins in 0.5% (w/v) Ponceau S in 1% acetic acid for 5 min, followed by destaining in distilled water for 2 to 3 min. After this time 1
To whom correspondence should be addressed at Department of Biochemistry, Midwestern University, 555 31st Street, Downers Grove, IL 60515. Fax: (630) 971-6414. E-mail: [email protected]
edu. 2 Abbreviation used: PVDF, polyvinylidene fluoride. Analytical Biochemistry 279, 241–242 (2000) doi:10.1006/abio.2000.4482 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
it is expected that protein bands will appear pink/red on a lighter background. Photography of stained membranes should be possible at this point in the protocol. Lanes are next cut out and placed in 7 ml of distilled water for destaining. With an additional 10 min of incubation in water the background is expected to be completely destained. Protein quantification is then done by measuring the absorbance (525 nm) of the eluted Ponceau S in each sample and calibrating to protein standards. In our experience using PVDF, the membranes became completely and irreversibly stained to a dark hot-pink/ red color following treatment with 0.5% Ponceau S. Protein bands could not be seen, although the presence of a wide range of molecular weight bands was observed on a duplicate gel stained with Coomassie blue. In addition, subsequent destaining proved impossible with water, or with various other solvents we employed (acetic acid, ethanol, methanol, and sodium hydroxide at concentrations ranging from 0.1 to 1%), even when destaining was attempted for periods up to 24 h at room temperature or at 37°C. The Ponceau S stain remained firmly affixed over the entire surface of the membranes. We were equally unsuccessful with PVDF membranes that contained newly transferred proteins as with those that had been processed as immunoblots (blocked, incubated with primary and secondary antibodies, and detected by chemiluminescence using standard methods). We modified the original procedure as follows. A PVDF membrane (9 cm ⫻ 12 cm, 45 m) containing proteins that were transferred from an SDS–polyacrylamide gel was rinsed briefly in distilled water and then soaked for 1 min in a 25-ml volume of a solution of 0.2% (w/v) Ponceau S (Sigma Chemical Co., St. Louis, MO) in 1% acetic acid. Protein bands in lanes were observed almost immediately. Excess, unbound stain was then removed by a brief rinse with distilled water. The membrane was next cut into strips containing individual lanes and placed inside 10-ml glass test tubes containing 7 ml of NaOH at concentrations ranging from 0.05 to 1 N. To determine the optimal destaining time we incubated Ponceau S-stained PVDF strips in the various concentrations of NaOH for times ranging from 30 s to 25 min. Destaining with 0.1 N NaOH for 5 min at room temperature caused complete elution of the Ponceau S; destaining with higher concentrations of NaOH and/or for times longer than 5 min did not increase the amount of eluted 241
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are shown (Fig. 1). Similar results were obtained whether or not the PVDF membrane had been blocked (in 3% nonfat dry milk in phosphate-buffered saline with 0.1% Tween 20 for 1 h at room temperature) prior to Ponceau S staining and/or treated as an immunoblot (incubation with primary and secondary antibody after blocking and prior to detection) prior to Ponceau S staining (data not shown). Therefore, this technique works well for the range of protein concentrations of cell lysates routinely used for immunoblotting applications. In conclusion, we have modified the Ponceau S staining procedure for use with PVDF membranes. This modified protocol provides for a rapid quantification of proteins on PVDF membranes and allows for verification of equal sample loading and uniform blotting efficiency, highlighting its usefulness in quantitative immunoblotting.
FIG. 1. Relationship of protein concentration and absorbance of eluants from Ponceau S-stained PVDF membrane. Cell lysates from murine thymuses were prepared as previously described (4). Protein concentrations were determined (Pierce Chemical Co., Rockford, IL) and appropriate volumes were loaded in individual wells of a 10% SDS–polyacrylamide gel to yield 1, 2.5, 5, and 10 g per lane. Duplicates of each sample were run. Following electrophoretic separation for 30 min on a mini gel (Mini PROTEAN II electrophoresis cell, Bio-Rad, Hercules, CA), proteins were transferred to a PVDF membrane (Micron Separations Inc., Fisher Scientific, Pittsburgh, PA) using a semidry blotter (Bio-Rad) and standard conditions. Membranes were then treated according to the modified Ponceau S staining protocol with absorbance at 525 nm determined for eluants from individual lanes. Absorbance at 525 nm for blank, negative control lanes (mean OD 525 nm ⫽ 0.5 ⫻ 10 ⫺4) was subtracted from the absorbance readings for each lane that contained protein. Results of two experiments with mean values of duplicate samples are plotted. Error bars represent standard errors of the mean.
Ponceau S as determined by absorbance reading at 525 nm. Eluants were transferred to cuvettes and their absorbances at 525 nm were determined in a spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). To ascertain whether this approach would be reliable for quantification of proteins that had been transferred to PVDF, we electrophoresed duplicate samples of cell lysates (4) of known protein concentration (ranging from 1 to 10 g/lane) on a 7.5% polyacrylamide gel. Next we transferred the proteins to PVDF, stained the membrane with 0.2% Ponceau S in 0.1% acetic acid for 1 min, cut the membrane into strips containing one lane each, eluted the Ponceau S by incubating in 0.1 N NaOH for 5 min, and read the absorbances of the eluants at 525 nm. Two lanes from the gel in which loading dye only and no protein samples were electrophoresed were included as negative controls; absorbance at 525 nm of the eluants from the negative controls was subtracted from absorbance of lanes containing proteins. Results from two independent experiments with duplicate samples in each
REFERENCES 1. Salinovich, O., and Montelaro, R. C. (1986) Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Anal. Biochem. 156, 341–347. 2. Klein, D., Kern, R. M., and Sokol, R. Z. (1995) A method for quantification and correction of proteins after transfer to immobilization membranes. Biochem. Mol. Biol. Int. 36, 59 – 66. 3. Current Protocols in Immunology (1998) (Coligan, J. E., Kruisbeek, A. M., Marguiles, D. H., Shevach, E. M., and Strober, W., Eds.), Section 8.10.8, Wiley, New York. 4. Viselli, S. M., Olsen, N. J., Shults, K., Stelzer, G., and Kovacs, W. J. (1995) Immunochemical and flow cytometric analysis of androgen receptor expression in thymocytes. Mol. Cell. Endocrinol. 109, 19 –26.
Native Extraction of PhosphotyrosineContaining Proteins: Requirement of Tyrosine Kinase Inhibitors to Obtain Specific Phosphorylation Signals Manfred Johannsen,* Martin Ru¨hl,* Dirk Manski,* Rajan Somasundaram,* Ernst Otto Riecken,* and Detlef Schuppan† *Department of Medicine I, Klinikum B. Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany; and †Department of Medicine I, University of Erlangen-Nuernberg, Krankenhausstraße 12, 91054 Erlangen, Germany Received September 20, 1999
Tyrosine phosphorylation of cellular proteins and activation of protein kinases are well-known phenomAnalytical Biochemistry 279, 242–245 (2000) doi:10.1006/abio.1999.4473 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.