Hallo,
I am doing some western blot experiments at the moment and have some problems. I transfected a construct in HEK293 cells and loaded a lysate to sds gel and blotted the proteins to nitrocellulose membrane afterwards. Our group generated some new antibodies against the appropriate protein and we wanted to validate the antibodies in western blots. I have another commercial antibody as well that should work in western blots, however I don't get any signal for that in the HEK cell lysate. In cell lysate from vsmc, where this protein is endogenous available, it works however fine.
Do any of you know reasons, why the antibody works not for transfected protein?
It is either a transfection or construct issue. You got any marker on your plasmid that demonstrates the successful transfection into 293 cells?
Would you like to give more detail on
- How do you lyse the cell and get the lysate? Did you add sufficient proteinase inhibitor?
- Have you try to stain the trial run gel with coomassie stain? It will be veey helpful to check the efficiency of the electrophoresis of the protein?
- How long have you block the membrane? What blocking solution are you use?
@ Syed A Ali: I transfected the construct together with another construct containing m-cherry. I checked the transfection sucess with fluorescence microscope and it appears to be sucessfull.
@ Wai Chin Chong: I lysed the cells with a nonidet-p-40 cell lysis buffer. I usually wash the cell with PBS before. Then I add lysis buffer and leave the cells sitting in the fridge for 5-10 minutes. When the cells are detaching from the surface I can harvest them and centrifuge to remove the buffer. Afterwards the cells were washed several times with pbs. I add sufficient amounts of fresh proteinase inhibitor to the buffer before lysis.
I didn't do a coomassie stain but I stained the blot with ponceau s and got a lot of protein bands, also for bigger proteins as mine.
For blocking i use a 5% milk solution in 1XTBST and block the membrane for 1 h at room temperature.
As Syed A Ali mentioned before, it seems like you encounter a construct issue.
Transfecting another vector containing m-cherry is not a good control :
- First, you decrease the probability that your cells integrate your plasmid of interest (competition)
- Second, if it is not the same backbone, with the same promoter etc... It is meaningless.
What i do as often as it is possible is putting a tag in my construct (flag, HA...) so i can see it in WB and immunoprecipitate it easily. Or at least a GFP in my plasmid.
Good luck
Thank you for your answer david.
We had a construct with a gfp tag before, however the gfp got a mutation and so we weren't able to confirm transfection. At the moment we don't have a construct with a fluorescent tag, that's why we used a construct with m-cherry, but we trasnfected it in a ratio of 1:15 to the other construct. So I quess that it is more likely that the construct were taken up as well, when m-cherry were taken up.
Do you think it's possibly anyway, that the construct were NOT transfected in the cell?
if you can detect same protein in other cell line with the same procedure then this might be an transfection or construct issue
I agree on @ Aditi. The western protocol seems fine. It is more likely to be transfection or construct issue.
I don't think your transfection failed, given the ratio you gave.
I would go for a construct issue, so be sure of the sequence and if possible, subclone a GFP or tag.
As others said, transfected cells are not expressing your protein of interest.
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