Hi,
in order to help and give you some tips and pointers, it would be good to know what exactly you have tried and what you are having problems with. Do you get any bands at all, what are your controls, do you have detection problems, etc. Have you tried your protocol with cell lines or similar (ie not stem cells)? With that info we may be able to point you into the right direction.
Best
Erik
Thanks for your interest dear. Actually the main problem was the background although i can see the bands but not so strong and also background is not uniform like others. And also at the same time in same gel and same membrane, i can clearly see the Actin band . I changed the antibody for LC3, changed membrane, also tried different cell lines but the problem remain same. You can check the attach files
Hi,
sorry for the late reply, last week flew by. Actin is supposed to be strong, that's why they use it as a control. Is that band from a positive control or from normal cells? LC3 Antibodies should yield 2 bands (LC3 I & LC3 II). So first make sure you dont use an antibody that is specific to one version (not sure if they exist, but always good to exclude that). As a positive control you can use Rapamycin. You probably have to try different concentrations and incubation times for your cells. If you have the right antibody and control and still don't see any difference, you can tweak the detection:
From your pictures it looks like you are using two different detection methods (ECL solution for LC3 and some light emission assay for actin?) Why dont you use the same for both?
If you do, you can try to boost the LC3 signal, by using a biotinylated conjugate. So you would use your Anti-LC-3 to bind to the membrane and then use a secondary antibody that has biotin instead of HRP (this way you dont need to buy an LC3 antibody with biotin conjugates). then you add HRP conjugated to Strepdavidin. Since Biotin and Strepdavidin are smaller than Antibodies and bind very stronly to each other, each antibody can bind several HRP-Strepdavidin molecules which then can process more ECL. Hope this is giving you something to work with.
Thank you so much for your time. I will try all those which you suggest for me.
These bands are from normal cells, I use antibody which is specific to both LC3I and LC3II.
I use same detection method for both and also these both are from same gel and same membrane
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