Lysis buffer composition dramatically affects extraction of phosphotyrosine-containing proteins.

The oncogene v-src encodes a protein tyrosine kinase that can transform rodent and avian cells to a fully neoplastic state (1). As part of our analysis of mutant v-src alleles encoding proteins with reduced kinase activity (6,7), several different lysis buffers were used. While comparing protein blots probed with anti-phosphotyrosine antibodies, it was found that the specific phosphotyrosine-containing proteins observed depended not only on the allele being expressed (as expected), but also on the buffer in which the cells were lysed and the particular anti-phosphotyrosine antibody used. Three transforming alleles and one nontransforming allele were used in this study. The identities of the alleles and their relative kinase activities are indicated in the legend to Figure 1. Cells expressing various v-src alleles and cells that were mock transfected or vector-alone transfected were washed three times in cold phosphate-buffered saline (PBS) made by The Pennsylvania State University core facility: 116 mM NaCl, 12 mM Na2HPO4, 1.5 mM KH2PO4. They were then lysed on ice in the three different lysis buffers: A (50 mM Tris-HCl [Sigma Chemical, St. Louis, MO, USA], pH 7.5, 150 mM NaCl [Fisher Scientific, Pittsburgh, PA, USA], 1% Nonidet® P-40 [NP40; Sigma Chemical], 0.25% Na+ deoxycholate [Fisher Scientific] and 10 μg/mL bovine serum albumin [Sigma Chemical]); B (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 50 mM NaF [Sigma Chemical], 30 mM Na4P2O7 [EM Scientific, Cherry Hill, NJ, USA], 150 mM Na3VO4 [EM Scientific], 5 mM EDTA [Sigma Chemical] and 1% Triton® X100 [Sigma Chemical]); and C (30 mM Tris-HCl, pH 6.8, 150 mM NaCl, 1% NP40, 0.5% Na+ deoxycholate and 0.1% sodium dodecyl sulfate [SDS] [Calbiochem-Novabiochem, San Diego, CA, USA]). All three lysis buffers were supplemented with 300 μg/mL phenylmethysulfonyl fluoride, 20 μg/mL aprotinin, 10 μg/mL leupeptin (all from Sigma Chemical) and 100 mM Na3VO4. Proteins from equal amounts of cell extract were separated on 10% polyacrylamide gels containing SDS, blotted to nitrocellulose and probed with three different anti-phosphotyrosine antibodies (α-Ptyrjw from Jean Wang of UCSD, San Diego, CA, USA, FB2 from CRL 1891 cells from ATCC [Rockville, MD, USA] and 4G10 from Deborah Morrison of NCI, Frederick, MD, USA). Because the anti-phosphotyrosine antibodies were made to different phosphotyrosine-containing sequences, it was expected that the antibodies would detect different phosphotyrosine-containing proteins, and this was the case (see Figure 1). For example, phosphotyrosine-containing proteins of a variety of sizes were detected in all cells with 4G10, whereas FB2 strongly detected bands at approximately 45 kDa and approximately 66 kDa, and αPtyrjw strongly detected bands at approximately 45, 66 and 97 kDa. With all lysis buffers, an increase in