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What are the suggestions for troubleshooting TAP western blots?
There are several things to consider with western blots for TAP-tagged yeast strains:
1) Was a loading standard for the western used to ensure the same amount of protein in each well? Too much protein can cause background.
2) Consider that there can be post-transcriptional splicing (producing smaller band than expected); multiple translational start point (producing multiple bands); post-translational processing (multiple bands); aggregation of expressed TAP-tagged proteins making dimers and trimers (producing bigger band than expected).
3) Are protease inhibitors being used in the extraction protocols (especially if the protein is particularly prone to lysis)?
4) Is it possible that contamination is being introduced anywhere in the process?
5) Dimerization can be occurring. Increasing stringency may help.