Repeated probing of western blots obtained from coomassie brilliant blue-stained or unstained polyacrylamide gels.

For several reasons, it can be desirable to probe a filter obtained after Western blotting of polyacrylamide gels with different antibodies. On the one hand, protein amounts may be limiting and re-probing of a protein filter may be the only way to identify different antigens. On the other hand, in the case of two-dimensional (2-D) gel electrophoresis, exact reproducibility of the protein pattern is difficult, and reusing the same blot ensures reliable comparison among different polypeptides using the appropiate antibodies. We have developed a method to reprobe polyvinylidene fluoride (PVDF) membrane blots obtained by electrophoretic transfer of proteins separated by either one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or by 2-D gel electrophoresis (isoelectric focusing [IEF], SDS-PAGE) up to 5 times. For detection of the antibody complexes, horseradish peroxidase-coupled secondary antibody and the enhanced chemiluminescence (ECL) detection system from Amersham International (Braunschweig, Germany) were used. Four of the five antibodies used for this study were raised in rabbits, and one was raised in mouse. The first antibody was raised against maize rpoA plastid gene product over-expressed in E. coli. This protein is a 38-kDa subunit of the soluble RNA polymerase encoded on the plastid DNA of higher plants. Two more antibodies raised in rabbit are specific for the αand the β-subunit of plastid ATPase purified from spinach plastids (3). Their molecular weights are 55 and 57 kDa, respectively. The fourth antibody was raised against a synthetic polypeptide homologous to a 158-kDa protein purified from barley plastids. It reacts with both a 158and a 154-kDa protein. Depending on the age of the plastids, one or two bands can be detected. An anti-α-tubulin antibody was purchased from Sigma Chemical (Munich, Germany). This antibody, which was raised in mouse, reacts with a 50-kDa protein. Protein samples analyzed were derived from two types of barley plastids: mature chloroplasts or young chloroplasts enriched in proplastids (“proplastids”). Isolated intact plastids were disrupted in 50 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 25% (wt/vol) sucrose, 50% (vol/vol) glycerol, 0.2 M ammonium sulfate and 2 mM dithiothreitol (DTT) using a glass homogenizer. Centrifugation at 180 000× g for 3 h at 4°C yielded two fractions: a soluble fraction and a membrane fraction. The membrane fraction was used to prepare a transcriptionally active lysate according to Rushlow et al. (7). The soluble protein fraction was purified by diethylaminoethyl (DEAE) anion-exchange chromatography (2). Protein-containing fractions were collected, combined and further purified by heparin-sepharose affinity chromatography (5). Fractions containing soluble RNA polymerase activity were collected and used for gel electrophoretic analysis. Fractions of membrane or soluble proteins were boiled in SDS sample buffer and separated on a 7.5% SDSPAGE (4). Two identical gels were run in a twin gel chamber (Biotec-Fischer, Reiskirchen, Germany) overnight at 16 mA. Proteins on one of the gels were silver stained (1). The second gel was equilibrated in 20 mM Tris, 150 mM glycine and 0.1% (wt/vol) SDS (cathode buffer) for 15 min and then trans-