Dear all,
I generated an antibody against a quite hydrophobic peptide of a protein. The immunization worked and it is possible to detect the specific peptide in an ELISA. It is also possible to detect the purified protein (50 ng from procaryotes and eucaryotes) specifically on a Western-Blot (see figure labeled with GST) or a Dot-Blot. Unfortunately using a complex lysate (30 µg) with strongly overexpressed protein just leads to a huge amount of background bands (see EGFP and FLAG-V5).
I already tried to modify the amount of detergent from 0,1 % - 1,0% of Tween 20 or Triton-X100. Both does not increase specificity of the signals.
Do you have any idea how to get a specific and robust signal?
Best regards,
Daniel
Is that protein modified in the cell? We've seen among some overexpression experiments where the protein is over expressed but we get one band instead of all of the modified bands? Maybe this is the issue to why it's not binding to the overexpressed protein of interest? Did you try changing your blocking buffer or your detergent( In respect that if your antibody binds too weakly, the tween in your buffer that normally prevents non specific binding could be preventing the antibody from binding to the protein, we see it with the ECRG2 tumor suppressor. Sorry I couldn't be more of help.
Thank you for your reply! I also thought about modifications. But the antibody also recognizes purified proteins from eucaryotes that should be modified the same way as in the lysate.
Regarding the detergent, I tried Tween 20 and Triton-X100 in concentrations between 0,1 and 1,0%. It did not change the band pattern in the lysates but Triton-X100 is leading to a stronger signal for the purified proteins compared to Tween 20.
Hi Vanessa,
thank you for your answer! I quess the lysis is not a problem, I am using RIPA buffer (containing 10% Glycerol) successfully since years and the protein can easily be detected with an antibody against an other epitope. Just this specific epitope makes problems. But yes, I could try to lyse the cells (e.g. sonication) in the elution buffer of the purified protein to see if this makes a problem.
Best,
Daniel
Hi Daniel,
Did you perform centifugation after lysis? And possibly loose some precipitated protein in the pellet. Kind of inclusion bodies? In this case, sonication and stonger detergents as SDS should be used for better solubilization.
Hi Patrick,
thank you for your comment. Sure I pelleted the lysed cells. In my usual WB protocol I don't use SDS but LDS which is contained in the loading buffer and the samples are denatured before loading. I also thought about losing a part of the protein as it is known to aggregate, but as the normal antibody can recognize it easily - and in the correct size - there seems to be enough soluble protein left.
Best, Daniel
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