Please note the attached gel. The three antibodies are not the same but are working well recently in others hands. My question is why the band at around 17kDa looks blurry and smearing? That is supposed to be LC3 and is expected to look like 2 close but clear doublet with different intensity. I have striped and probed the same blot with another tube of LC3 from another company and gotten the same result.
One error I could recall is the electrode running buffer. I have mistaken the transfer buffer (Tris-Glycine without SDS) as the running buffer (Tris-Glycine with SDS) and used as the inner buffer for the run, whilst the outer buffer is reused Tris-Glycine with SDS so it was correct. I caught the error in the middle of the run and replaced the wrong buffer with fresh Tris-Glycine with SDS buffer.
Tris-Glycine SDS PAGE, 12% Tris-SDS gel, loading buffer with SDS and 2-mercap, sample-buffer mixture boiled at 100C for 5 mins.
Resolution obtained is dependent on the % gel used. You could always try a 15% gel to see if you get a sharper band or opt for a gradient gel.
Sometimes, expired or not fresh reagents of a detection method (e.g. ECL) could cause this problem.
Low molecular weight requires high percentage gel concentartion i.e molecular weight of samples is inversely proportional to gel concentartion in SDS page
Dear Tsemay, better you can try in 15 or 18% gel and with 35, 40, 45 and 50 ug of protein. If band comes then go with good combination in which resolution of band is higher. Allow the gel to run up to end. Please stick up to staining. Standardize it properly. If band comes then go for transfer. If band is not coming then there may be problem with extraction of protein of interest you are looking for. Accordingly try to extract your desired protein in some other method. Reduce the time to 95 degree C with 4 minutes.
I agree with the recommendation to use a higher percentage acrylamide as a first option. Anything that diminishes the retention factor of the bands should decrease smearing. Another option to try - though I would try it secondarily - is to alter the ratio of acrylamide to bisacrylamide such that I had more frequent crosslinkages. You don't say what your ratio is, but if you use 19:1 A:B, your retention factor will be less than if you use 29:1 A:B.
Here is a protocol for detecting small proteins: https://www.lifetein.com/Detect_Small_peptide.html
And tricks on Western blot:
https://www.lifetein.com/chat/084013-Western-Blot-Technique-Theory-and-Trouble-Shooting
Thank you for all the answers. So after all the trying, the fix to my particular case is to run it slower. (same condition, same antibody, everything the same as the original post EXCEPT I ran this gel at 50V for 30 minutes and 80V for the rest of the gel) (this picture is showing two cut blots at the same molecular weight range and probed with the same antibody as above)
Tsemay Tse I've been having this exact problem. What gel system do you use? I've tried running my gels at 70v but it takes a very very long time. How long does it take you to run your gels at 80V?
Hi Aisling, I cast the gel and prepare the buffer myself using this protocol.
http://ecoliwiki.net/colipedia/index.php/Methods:Tris-Glycine_PAGE
it took me 2.5 to 3 hours to complete the run for a 12% gel. If nothing works for you you can try to make fresh APS from powder, use fresh bottle of acrylamide, and use more TEMED if it appears yellow with white precipitate accumulated at the bottom (oxidized TEMED under suboptimum storage - I used 3 times more for the resolving gel - and you don't need to change the % of TEMED for the stacking gel) the point for this is that to optimize the polymerization time - can't be too fast or too slow. You would expect the gel comes to full set in 10-15 minutes.
all the best to your experiment.
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