I'm studying autophagy in human podocytes (glomerular epithelial cells) and using rabbit polyclonal LC3B antibody from Sigma. I have been studying the optimal rapamycin concentration to induce autophagy, and found out that the antibody detects THREE bands of about ~18 kDa, ~16 kDa and ~12 kDa in size. Previously it has been shown that podocytes exhibit a high basal autophagy level (high level of LC-II even without autophagy induction). The problem is that now I don't know which band is the correct LC3-II. The middle band has the weakest intensity but it is correct in size. Its intensity also increases with rapamycin treatment. Sigma's antibody is rabbit polyclonal and made against amino acids 2-15. Thus it's different from LC3A.
I lysed cells in 1% NP-40 buffer (20 mM Hepes, 150 mM NaCl, pH 7.5), run 40ug on 15% gel (70-100V), blotted to Immobilon-FL membrane (PDVF) either 200mA for 2h or 55mA for 16h, and incubated with primary antibody overnight (1:3000). Here's also a picture on by blot (the standard band in the middle is 15 kDa).
Could anyone help me in interpreting these bands?
Just found out that Mizushimas and Yoshimoris review on LC3 interpretetation said that LC-II is usually 14 kDa and LC-I 16 kDa. Do they mean LC3B or LC3A, or does it make any difference?
Hi, I've already ordered bafilomycin but haven't received it yet. That's true, that should tell me which band is accumulating. But do you agree that usually LC-II is around 16 kDa? Unfortunately, very few publications show the protein ladder on the blot.
I use Odyssey infra-red imaging system so I take a direct image on the blot. It is a very sensitive system - that may explain the third band that is not usually detected with other imaging systems. In this paper the third band has been shown: http://www.ncbi.nlm.nih.gov/pubmed/?term=Guidelines+for+the+and+interpretation+of+assays+for+monitoring+autophagy So it may be an intermediate product on LC-II. But still it remains unknown why these strong bands are not 16 and 18 kDa...
Hi,
bafilomycin greatly increases the intensity of 14 kDa band so it seems to be LC-II. Probably I'll try also another antibody.
Perhaps this may be Pro-LC3 which was just translated before being cutted by ATG4B. A plos one paper used MS to prove that the top band is LC3-I, the middle one is Pro-LC3, and the bottom band is LC3-II.
Here is the doi: 10.1371/journal.pone.0074222
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