Polyacrylamide gel electrophoresis
Relative to protein standards, the apparent molecular weight of bR is 25,000 while the apparent molecular weights of pR-wt and pR-TCM are 36,000 and 31,000, respectively (fig. 2, lanes E and C, respectively). SDS-PAGE (fig. 2) also confirms the estimates of purity level based on the assumed ε280/ε546 ratio identical with that of detergent solubilized bR. Interestingly, the pR appears to be a doublet band whose relative concentrations remain almost unchanged during purification. This doublet is also present in the less-purified sample of pR-TCM, with both bands shifted down by approximately the same amount (fig. 2, lane C).
Figure 2  SDS-PAGE of pR (wild type and pR-triple cysteine mutant). Lane A contains bacteriorhodopsin (bR). Lanes B and F contain BioRad protein molecular weight markers including labeled bands at 21.5 (trypsin inhibitor), 31 (bovine carbonic anhydrase), and 45 (ovalbumin) kDa. Lane C is of the pR triple cysteine mutant (TCM). Lane D contains the Phenylsepharose™-purified pR wild type protein, corresponding to spectrum B of fig. 1. Lane E contains the hydroxylapatite-purified pR wild type protein, corresponding to spectrum C in fig. 1. Subsequent SDS-PAGE analysis of pR samples that had been stored for periods of time up to several months indicate that after sitting for several weeks in octylglucoside solution at 4°C, the largest post-translational modification on wild-type pR is eliminated – presumably hydrolysed off of the cysteine(s) – leaving only a 31,000-MW band indistinguishable from that seen for pR-TCM (data not shown). Furthermore, after boiling for several min in gel loading solution, this cleaved wild-type protein, as well as the TCM, both give an extra artifactual band near 36,000 dalton. The latter band, a singlet, is coincidentally at almost the same apparent MW as the doublet from the uncleaved post-translationally-modified wild type pR (fig. 2, lane E). These potential artifacts should be taken into consideration in any attempt to reproduce the results in Fig. 2.