Hi guys,
I tried to express my protein of interest in A549 cells which has an internal Flag tag. However, I can see multiple bands on my western blot after anti-Flag development. They run at different molecular weights and the expected one is also obtained. I can't figure out why I got multiple bands especially because the blot looks clean and it doesn't look like there is a lot of background. I was thinking about proteolytic processing... Are there some useful databases to check that? How can I find out which proteases occur in A549 cells?
I already sequenced the plasmid but it looks completely fine.
I appreciate your help.
Cheers, Lena.
Why do you say the blot looks clean if you are seeing multiple bands instead of just one?
Bands below the expected one could be due to proteolysis. The cells contain proteases, of course, especially in lysosomes. When you lyse the cells, you should include a protease inhibitor cocktail.
standardization of western blotting
example: try different conc of antibodies
washing of blotting paper with wash buffer to remove week interacted antibodies.
incubation time.
substrate concentration.
the multiple bands could be due to proteolysis ro degraded
Adam B Shapiro because there is no background and the signal looks very specific...
Thanks for your answers!
It can either be proteolysis (as correctly suggested before already) or premature ending of translation by the ribosome (although I find this less likely). The latter you will only see of course, if your tag is at the N-terminus. Where is your Flag tag located? N- or C-terminal?
If C-terminal, your translation must have gone to completion and it's probably proteolysis.
Do you see multiple bands during expression already (i.e. you directly lyse cells in sample buffer and run a WB)? Or only upon mild lysis, purification etc? The first would indicate proteolytic processing during or direct after expression (this will be hard to prevent, I think), the latter would point to protease cleavages e.g. by mixing different compartments and a protease inhibitor cocktail could be a possible solution.
Steven Verhelst the FLAG tag is internal although not disrupting the proteins secondary structure (it was not possible at N- or C-terminus due to signal peptide and interaction domains). I see this multiple bands in the expression test and the pull-down experiment although I added protease inhibitors. Also, the mass-spec experiment with the pull down experiment did not detect the bait protein... If I develop the blot with an antibody against the protein itself I can see four additional bands. Three run lower and one higher (20 kDa more).
Strange that you see additional bands of your protein without the FLAG tag, because the tag is internal. This would suggest multiple proteolytic processing steps that eliminate the FLAg tag, but it would not explain the higher band of course. Is it known whether the enodgenous protein is proteolytically processed? You may have already tried alternative expression hosts? If the goal is to obtain pure full length protein, you may need to go to a cell-free expression system, but you would need to ask someone else for advice on that, as I have very little experience with that.
This webtool by the labs of Rob Pike and James Whisstock may be of interest to you https://prosper.erc.monash.edu.au/home.html
Steven Verhelst I only see one band for the endogenous protein however, I can not exclude whether the epitope has been degraded or not... Thanks for your help! I transfected other cells and hopefully will see a clearer result.
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