Hello, I have set up the western blot in my new laboratory, but I can not make any bands.
All buffers and materials are new except film (within the expiry date without opening). I conducted Ponceau S staining to check whether the transfer was well (=transfer well). And I checked interaction between 2nd Ab and ECL with simple dot blotting of the Ab (=work well, I decided the dilution factor of the Ab).
I blocked the membrane with 5 % skim milk for 1 hour and probed GAPDH Ab (1:500, santacruz) in 5 % skim milk O/N. And I probed the 2nd Ab (1:3000, CST) in also 5 % skim milk for 1 hour and exposed film for 1 min. But there is no band (even any non specific bands or background signal).
Then, what can I do for next to check my protocol? Make me set priorities.
Did you check the source of 1st antibody?
And did you use correct source of 2nd antibody?
What is your 5% skim milk prepared in? It should be TBST or PBST buffer.
Yes, I saw the clear bands of proteins on membrane.
Actually, I did Ponceau S staining after film exposure because of the bad result.
Did you check the source of 1st antibody?
And did you use correct source of 2nd antibody?
Check the source of your primary and make sure your secondary is against this species. Has anyone in your lab used this primary before? Is your secondary HRP-linked? And what is your ECL reagent? You can check its efficacy by adding a couple of ul of secondary in 1 mL or so of your ECL solution; it should glow in the darkroom. You may also need to expose the film longer- 5 or ten minutes or even more (although GAPDH should be a decent band without a long exposure).
It would help troubleshooting if you gave further details, including sample type, lysis protocol, amount of protein loaded etc.
Julia, thank you for your advice. I will do it.
Do-wan, I checked, but there is no problem in source of Ab. I used rabbit IgG for all 1st Ab having correct cross-reactivity (Human, Mouse). And I used 2nd Ab for Anti rabbit IgG HRP linked Ab. Thank you for your advice.
Diana, all is new. so, nobody used 1st Ab before. I want to check the 1st Ab activity and interaction with 2nd Ab but I don't know how. I checked the interaction of ECL and 2nd Ab. I used commercial ECL and it is a new one. Then, I will increase the exposure time first.
My sample was B16F10 cells (mice) and I used commercial RIPA buffer for lysis with protease inhibitor cocktail obtained from Sigma. Thank you for your advice.
To check that all is working, I would cut the blot well above the size of GAPDH, and probe the top half with an antibody that you or someone else has already used in the lab (preferably one that uses the same secondary antibody as the GAPDH). This should be known to work, and therefore if it doesn't it tells you there is something wrong with the transfer or some of the reagents - and then you can do some further troubleshooting to find out what.
If that works but GAPDH doesn't, then it is possible you've got a bad batch of antibody. GAPDH is very abundant, so it shouldn't be difficult to detect; if it continues to not work I would contact the supplier and ask them to replace what they've sent you.
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