I'm doing a denaturing western blot on tissue lysates and cell lysates. I have an antibody that seems to work on the former (shows a band only in tissues where it is known to be expressed), but not on the latter (no change from wild-type to transfected cells).
- I'm transfecting the cells with a plasmid containing the construct of the protein of interest fused to a C-terminal GFP tag. The antibody is directed towards a N-terminal region of the protein.
- The cells are HEK, transfected with Lipofectamine, and the GFP fluorescence was clearly visible (no doubt of autofluorescence) and abundant.
- Tissues and cells are lysed with the same buffer (I've tried both 4% SDS-PBS and various versions of RIPA, the latest containing also CHAPS and SDS).
My next check will be blotting with a GFP antibody, but I mean, the green was there. I could also sequence the plasmids, but I got the correct sequence from the same company before.
Hello Iris Villani
If your antibody is able to detect the protein in tissue lysates, then it should detect the same in transfected cells too. Your transfection was also successful since you've seen green fluorescence in your HEK cells. In my opinion, there are very few possibilities. (1) Either your plasmid sequence is not in frame leading to translation of an entirely different protein that lacks the epitope for binding to your antibody. (2) The plasmid that you are using for transfection lacks your cloned gene (very least likely) since you're not able to detect the protein. (3) Your protein may have entered into the insoluble fraction (could have been wrongly translocated). After lysing your cells, you may visualize the fluorescence in the pellet (cell debris) and the supernatant. Most probably, it is something to do with your cloned construct. Hopefully, you are able to identify the issue. Good luck!
Dear Iris Villani
To the valid possibilities listed by Jatin Chadha I would add the probable difference in posttranslational modifications of the protein in HEK cells and the tissue.
Hi Iris,
We at Xenotox, Inc. (http://www.xenobiovir.com/) have had the very same problem you described but regarding type A influenza virus nonstructural protein 1 binding protein (NS1BP). Lysis refers to the breaking down of the cell thus compromising its integrity. So, with specific to NS1BP antibody it was shown a band only in tissues lysate, but not on the cell lysate. I happened until we started using SoluLyse-M™ Mammalian Protein Extraction Reagent (a novel nonionic detergent designed for efficient whole-cell protein extraction from cultured mammalian cells). It gently and rapidly dissolves cell membranes at low concentrations without denaturing proteins (and there is no need for secondary treatment such as sonication or freeze-thaw).
Good luck,
Ilya
You should keep in mind that the antibody you used (mono or polyclonal?) is detecting an other protein with the same molecular weight; this could happen with commercial antibodies that are not checked very carefully (if they detect a band in the right MW they are OK to be sold) if you can have a cell line with your GOI knocked down it will be a good control. more over the fusion of the GFP tag to your protein can modify the affinity of your antibody for the fusion protein (especially if you use a monoclonal)
the GFP western will tell you what happen
let us know...
Hello everyone and sorry for the delay.
Here is my blot with an antibody targeting the GFP tag.
wt= non-transfected; R and S = ripa and sds lysis buffers; day1/2 = harvest time (after transfection). The band about 17 kDa is a loading control.
1) the protein is definitely expressed in the transfected samples.
2) it is a smear towards lower MW, plus a couple more isolated bands at even lower MW.
3) it is all below the expected size (50 to 70 instead of 80),
4) in the blot with anti-protein AB (which is polyclonal), nothing is detected in this range.
So, I would think that the cells are degrading the protein starting from the N-terminus. This makes sense both because the anti-protein AB is directed against that part, and because the GFP is attached on the C-term and it seems still recognised.
I also transfected another protein (a citosolic one, while this is a membrane transporter), with a different tag, and it showed up as a neat clean band at the expected height. So it's not a problem of my lysis, nor my cells going berserk.
...I guess I'll use a tissue as positive control, but it would be nice to have my protein functional.
Any ideas? Didier Poncet ?
Hi,
probably the fusion with GFP destabilizes your protein (or impairs binding to another protein that form complex with your membrane transporter) which start to be degraded. can you try a Nterm fusion or a smaller tag?
at least from your gfp blot it is not a pb of antibody
good luck
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