Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types...

The most effective protein purification method of low picomole amounts for sequence analysis involves polyacrylamide gel electrophoresis followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. Since a critical factor in this procedure is the protein recovery at the blotting step, different types of PVDF membranes were systematically evaluated for their ability to bind proteins during electrotransfer. Differences in electroblotting recoveries occurred between types of PVDF membranes for some proteins. Some variability persisted even when optimized electroblotting procedures were used which reduce the sodium dodecyl sulfate (SDS) concentration in the gel and improve protein-PVDF binding. The membranes which were evaluated could be grouped as either "high retention" membranes (ProBlott, Trans-Blot, and Immobilon-PSQ) or "low retention" membranes (Immobilon-P and Westran). The high retention membranes showed higher protein recoveries under most conditions tested, especially for small proteins or peptides. These high retention membranes were also less sensitive to the exact electroblotting conditions, especially those factors which affect the amount of SDS present during either electrotransfer or direct adsorption from protein solutions. High retention PVDF membranes are therefore preferred in most cases for optimal protein or peptide recovery prior to direct sequence analysis. In contrast, low retention membranes are preferred for procedures where subsequent extraction of the proteins from the membranes is required. Even under identical conditions, substantial protein-to-protein variation for both adsorption and subsequent extraction is routinely observed for both groups of membranes, indicating that the nature of protein-PVDF interactions is more complex than simple hydrophobic interactions.