My lab is currently expressing secreted protein inside sf9 cell, with his tag for further purification. But during P2 western analysis, the polyhis antibody cannot detect but the protein antibody can detect inside the cell (it should be secreted). What happened to the histag, is the tag removed by the cell?
The tag may have been lost due to proteolysis, but another possibility is that your anti-His antibody is poor. Some of them are not very good. Try an antibody from a different supplier. Make sure you have a positive control for the Western.
If you have an antibody against the protein, perhaps you can use that for the Western.
The anti-His antibodies are not always reliable. Depends on protein folding and tag accebility. An alternative to Western is to actually test your protein mix directly on a small IMAC column. Your target proten should then bind and you can elute by a pH change or EDTA. You follow the fractions by SDS-PAGE. Good luck
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