I also have a problem with detecting the LC3II band on my western blot. The lC3I is not well separated from my LC3II band or the LC3II band is a smear.
I use 4-20%Tris/Glycine gels from Biorad. For the lysis step we use MPER extraction buffer with proteinase and phosphatase inhibitors. I use 75 Volt for the SDS-running, 35 Volt for the transfer time. I also use PVDF membranes. But I do not know if I should change the running, transfer buffer or the lysis buffer for the sample preparation?
What is the molecular weight of LC3II? How do you treat your samples before running?
Look at this article: http://www.ncbi.nlm.nih.gov/pubmed/17611390
Perhaps it can help you.
Hi Natascha, I also had/have problems with LC3BII. But I got some blots that gave nice results (my main problem are unspecific bands and a high background), I always use 15% homemade SDS-Page and run at 150V for 1:40 (miniprotean) and blot with 50V for 1 hour. I also tried gradient gels but did not get a good resolution. Since the proteins are so very small (16 and 18 kD) you have to run your gradient gel for a long time to get a decent resolution. Use prestained marker to estimate how far your protein has travelled in the gel.
Lc3II is a membrane protein so be sure to use a correct lysis buffer! I use a lysis buffer with 0,1% tritonX-100 and run in 13 or 15% polyacrylamide gel.
hi! the MW is 16 and 18 kDa.
I am harvesting the samples MPER extraction buffe mixed with proteinase and phosphatase inhibitors!!is this maybe not a good lysis buffer for LC3??
Which lysis buffer are you exactly using???
Hi Anika Stracke,
for SDS page i am using 75 or 100V for 1,30 min and for the transfer i used 35V and 1,40 min and this 4-20% tris-glycin gels from biorad.we are using self prepared running and transfer buffer.i am thinking if i schould change and try some ready to use buffers. which running and transfer buffer are you using??
tody i was imaging some LC3II blot again and i clud not detect the LC3II band.
I can't found the composition of MPER buffer. I never used it! can you explain what there is in it? I use a buffer containing 20 mM Tris (pH 7.5), 2 mM EDTA, 2 mM EGTA, 250 mM sucrose, 5 mM DTT, 200 mg/ml leupeptin, 10 mg/ml Trasylol, 1 mM PMSF, and 0.1% Triton X-100. I detect very well LC3 I and Lc3II in both cell and animal derived extracts.
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