I've been trying for months to detect my protein in Western Blot. Firstly there was an antibody problem, now I should have a perfectly good antibody which I want to test, but the protocol doesn't seem to be good enough.
My protein is 17kDa and is pretty delicate in a way that the antibodies recognize only a small moiety on this protein. Any advice on optimizing protocols for small proteins?
I know I should probably change the transfer: low voltage (eg. 20 V?) and longer time of transfer (eg over night). But also any other tips and hints? I want to eliminate the option of technical problems before I declare the antibody ungood.
Thank you for sharing your experiences, very appreciated! =)
If you suspect that your protein is lost somewhere along the western blot process, you may try dot blotting to test whether your antibody works in general. For smaller proteins in western blotting, there are some optimizations that can be done in addition to adjusting blotting time/voltage, such as fixation of the membrane after blotting or using optimized membranes with a smaller pore size. There is a useful handbook by MerckMillipore that goes into great detail about western blotting in general and contains a lot of useful information on handling smaller proteins as well:
https://www.researchgate.net/institution/Merck/post/Protein-Blotting-Handbook-Tips-Tricks-5dfa2ee9aa1f0994002800c5
One common example of a somewhat hard to detect small protein is alpha-synuclein. Here is a paper about improving it's detectability. It might also contain some interesting info for you!
Article Improved Immunodetection of Endogenous α-Synuclein
Hope this helps!
Cheers,
Jan
Jan Philipp Dobert Super, thank you, Jan!
Great book recommendation and thank you for the article. I'll have a look and compose a protocol. ^^
Agree with Jan Philipp about fixation. 0,5% PFA/PBS just after transfer (but remember to briefly wash the membrane from Tris - in PBS) for 30mins at RT. 3x washings - staining - washing - blocking etc...
The problem may be also that the epitopes are hidden - then try PVDF membrane instead of NC and vise versa. Another option is autoclaving the membrane just after transfer (you can find the protocol).
You can also try to enhance the signal by antibody biotinylation.
Good luck!
Aitsana Maslakova thank you! So, you mean right after the transfer: wash briefly with 1xPBST, fixating in 1x PBS for 30 min RT, then I block with 5% Milk in 1x PBST RT, 1h. Then the rest- primary AB, wash, secondary,...
I use PVD membrane 0,2 um pores =)
Anja, right after transfer I wash with 1xPBS, then fixation in 0,5% PFA..., wash in PBS 3x5mins shaking (be sure to add enough volume to get rid of PFA), then stain with ponceau S, than wash in PBST. You should also try BSA blocking (3-5% BSA in PBS 1h RT, shaking) as milk may hide epitopes. I used to have no signal on PVDF, but after I used NC membrane I saw the signal. Prepare 1 membrane with the same sample, cut the lanes and try any protocol in a parallel.
Do you have bands? Thick,thin,double? Are the bands unespecific?
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