I used to performed Western Blot to detect a protein with #14-16 kDa molecular weight (LC3) a few months ago. It worked very well. I have stopped doing that experiment for one month.
I am now doing it again with the same method but could not detect my protein after transferring onto PVDF 0.2um membrane. I used beta-actin as a loading control and it is still detectable, but not my 15kDa protein. Is there any suggestion? Thank you so much!
Probably it ran out. Use 15% gel and stop running before the dye reaches extreme bottom of gel.
Please mention ur procedure (gel%, transfer cndn etc) to let someone troubleshoot possible reason for inconsistency. U cannot detect bands at all or the conjugated lc3 is missing? Are u using old lysates? How were cells lysed?
Dear Goodwin and Manoj,
I used 12% gel for running step and stopped when the dye reached bottom of the gel, transferred to PVDF 0.2uM, 80V for 90min. All the bands of protein marker (Bio-rad) were observed on the membrane after transferring. I also tried with 15% gel, but the problem was not solved.
I could detect beta-actin but could not detect LC3I and LC3II.
is there any chance of that LC3 protein swims out from the membrane (because beta-actin is heavier so it still remained in the membrane)? Should I reduce the period of transfer step?
@Manoj: I kept the cell lysate at -70oC for 1 month and used again. IS it the problem? Cell were lysed in RIPA buffer including proteinase inhibitor cocktail.
Thank you very much!
Maybe, reducing the time of transfer may help and using 12% gel
You might want to add a bit of extra methanol and reduce the amount to SDS to your transfer buffer keeping the voltage and time of transfer same (70v/ 200ma for 90 mins). Block it for 2-3 hours ( if you are keeping it overnight). The idea is not to let your protein leak out of the blot ( sometimes happens due to prolonged blocking or high current or sometimes by methanol scarcity in used transfer buffers).
Dear Shoiab,
I used 20% Methanol for my transfer buffer. I though it was enough...In your experience, how many percentage of methanol you usually use?
Thanks again.
Dear Maryam,
Thank you for your suggestion.
I also changed my transfer buffer to a fresh one. I will try to exclude the SDS in transfer buffer and hope it works.
I am also looking forward to you book. Thank you in advance.
If it had earlier worked for you, you should try with some fresh lysates (may b lysed directly by boiling in loading dye) to make sure there's nothing wrong with your lysates. I have also observed inconsistent blotting results with old lysates for LC3. But it is normally with respect to the LC3-I and II ratio and some bands do appear.
Thank you Manoj,
I tried to use again the old lysate and the LC3 bands appeared like a charm. The only change I did is using another power supply. I could not believe that the power supply can cause such that problem to my western blot.
Thank you anyway for your help.
Like some of the IT discussion forums, may be researchgate needs an option for the thread initiators to add '' RESOLVED'' tag to their queries so that people dont follow discussing it for months after the real problem is solved.
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