I have created a plasmid for my protein of interested tagged with Flag tag. The sequence looks fine but for the life of me I cant get this protein to express itself and see it using western blot ( I even ended up buying the same plasmid from a company thinking it might just be something I did wrong) and still the same result. This protein is 34 kda and a membrane protein. Can it be that using RIPA* buffer isn’t good enough to see my protein via western blot? I feel like like even using Roig buffer I should still see some signal even if not much (this is an overexpressed protein at the end of the day) Any ideas?
Is there sufficient detergent to solubilize the membranes and release the membrane proteins? (I have to admit that I don't know what Roig buffer is nor what is in it - and google didn't help).
I would think that RIPA buffer should be sufficient. Are you doing this in E. coli? You might try and just solubilize the whole cells boiling in loading buffer and to see if your protein is present in the total cellular protein. That might help you distinguish between not making protein vs not detecting the protein in the membrane fraction. It is possible that your protein is just not inserting into the membrane and is remaining insoluble and being lost upon fractionation.
Can you give more information about the system you use for expression (E.coli, mamammlian or insect cells) and about the construct (e.g. where did you add the FLAG-Tag?). Keep in mind that expression of membrane proteins require a membrane targeting sequence on the N-terminus, that said. If you added your FLAG-Tag on the N-terminus, this protein will probably not be expressed anymore if you didnt put the membrane targeting sequence before the FLAG-Tag.
There are several possibilities:
1. Is your protein successfully expressed? Sometimes the overexpression of target protein may kill the cells and the cells collected for analysis do not express the target protein. You may try to do staining to verify this.
2. Except the buffer, did you boil your protein samples? For some membrane protein, you may not get any western blot signal if boiled 100C for 5 min due to aggregation. And for some membrane protein, it will change the moving rate of the protein in the gel. You may heat protein sample at 60C for 10 min, and check again.
Usually the N-terminus of a membrane protein sequence encodes a signal sequence for the cell machinery to incorporate this protein into the membrane. If you alter the natural N-terminus it could be that this protein is not incorporated in the membrane anymore. You either have to tag your protein on the C-terminus or have to add a membrane targeting sequence (e.g. like the HA-Tag) on the N-terminus.
That could be one explanation.
There could be other reasons though:
E.g. If you order the gene sequence, did you optimize the codon usage for the construct for the mammalian system you are using?
What antibody did you use? Against the FLAG tag or against the protein itself?
Hi Grace,
I can recommend the MP purification service provided by Creative Biostructure. I worked with them in the expression and purification of MP before. Creative Biostructure has a professional MP platform, and this website also has some resources for learning.
Hope it helps!
Your RIPA Lysis Buffer may be the problem here. Consider using a Urea Lysis Buffer instead.
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