Hi...
I expressed a scFv antibody in HB2151 e.coli by TES (Tris-EDTA-sucrose) buffer and now like to confirm it by Dot blot, however, I failed to visualized it using DAB.
I coat the scFv (with a C myc tag in its C-terminal) on PVDF membrane and incubate it 0.5 h at RT, block it by 3-5% of S.M (1 h at RT) then wash by PBST followed by PBS and finally add HRP-Conjugated anti-C myc antibody (1/100, 1/200, 1/400, 1/800, 1/100 dilutions). Then I add DAB for visualization. No results. The purified scFv also has a very sharp band on SDS-PAGE. by the way, I coat different amount of scFv antibody (3ul, 5 ul, 10 ul and 15 ul) on PVDF membrane. I checked the HRP part of the antibody directly by adding both DAB and TMB to it with 1/1000 dilution and it works fine.
Can I doubt the malfunction of binding property? I purchased the antibody from Roch company.
Hi Seyed,
Why are you doing a dot blot, and not a western blot against your antigen?
Also, have you sequenced the scFv and are you certain the tag is present?
I return to dot blot because the western blot had not any results. Yes, I sequenced my scFv and the C myc tag (EQKLISEEDL) is present in its C-terminal.
Do you have a positive control for your anti-myc antibody? Even if the HRP part works, the binding properties might be not working.
How did you do the Western? Do you get a lot of background or nothing at all? And I agree with Vivica, it will be good to have a positive control, i.e. a c-myc tagged protein that has worked before.
In our dot-blot we do not get background. Make sure that you let you proteins dry before blocking. We use 1%milk + 1%BSA in PBS for 1 hr.
Maybe a silly idea: You probably know that PVDF membranes have to be activated by soaking in methanol (not very healthy!) before use? This is at least necessary when used for western blotting but I guess it's the same for dot blots.
No I dont have positive control, and no background.
Yse I activate my paper by MeOH 15 min at RT.
Ok, I will try nitrocelluse type, too
Do you have another lot or at least a fresh aliquot of anti-myc? It sounds to me that your antibody might be the main problem.Maybe it might also help to dilute the secondary antibody much higher (1:5.000 / 1:10.000 /1:50.000 / 1:100.000) - which dilution does the supplier recommend? You should definitely try to find a positive control (myc-labelled protein). Another point: Have you tried to detect the protein on the membrane by Ponceau S staining or similar?
I agree with you on antibody problem as well as a positive control.
Our antibody is directed (HRP-conjugated anti c-myc antibody) thus no need for secondary antibody.
yes I wrote in my first question "a very sharp band on SDS-PAGE" using CBB staining.
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