Hello, researcher
I would like to ask about my western blot results
Last week, I performed dot-blot analysis on nitrocellulose membrane using mouse anti his 1:2500 (1st), and goat anti mousre IgG HRP and DAB substrate (sigma). I can detect positive results.
Now, I repeat the dot-blot again but using sing mouse anti his 1:1000 (1st) (new tube, same company), However, i get a negative result,In addition, the positive protein control also showed negative results.
So, i want to know Why?
[My dot-blot protocol 1. dot 2. block in 5% nfdm in TBST 1h 3. probe with mouse antihis 1:1000 over nigth 4 degree celcius 4. probe with goat anti mouse 1:5000 1h, rt 5. Add substrate mixture (DAB 3mg; sigma in 5ml TBS, 25ul of NiCl2 and 4ul of 30% H2O2)]
Thank you so much
Please repeat your assay at-least one more time without changing any condition and if the result comes out negative again, then perhaps we should look into it. Results (positive or negative) should be reproducible for meaningful discussion.
You could try a Ponceau staining in your membranes to check if there was a problem with the transfer. Once you know that your proteins are there, you could try a staining with an antibody that has always worked before in your lab (for example tubulin or actin) if it is avalaible for you to use. If it works, maybe the problem is in the new tube of anti-his.
I hope that it helps.
How many times did you wash the membrane after the overnight incubation with the primary antibody? It's very important to remove all the traces of the primary antibodies before applying HRP-conjugated secondary antibodies. That's because usually solution of primary antibodies contain sodium azide that is a potent inhibitor of HRP enzime. You should also pay attention to the volume of buffer that you are using in the washing steps to be sure to remove all the primary antibody.
I hope that it helps.
Syed A Ali above has the first, best advice.
Next, if the only parameter in your dot blots that you changed was the new tube of antibody and the higher concentration the second time, we have often seen new tubes of the same antibody with different lot numbers not working at all. Hopefully, if you repeat the dot-blot you can have parallel blots you can run identically except for the tubes of primary antibody. I have done this and the vendor has refunded our payment for the primary after providing them the data that the new lot didn't work--a case where we end users are the QC department of the company. Unfortunate, especially since experiments are often complicated enough to have to provide "bridge" experiments for newly acquired tubes of antibody.
From my experience it seems that sometimes the affinity of an anit-his6tag primary antibody denpends on the tagged target protein itself. It might be worth to try another primary antibody (from a different company).
Do you have any evidence that your target protein is in your sample?
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