LC3 antibody advice
Posted 12 January 2011 - 02:30 AM
Should i pick poly/mono-clonal? LC3 or the more specific LC3-B? are there any protocols available?
Posted 13 January 2011 - 06:25 AM
Posted 13 January 2011 - 07:13 AM
did you do a WB on human macrophages? would you mind posting a picture of your WB and sharing your protocol?
No, it was not on human macrophages. It was actually on homogenized human brain tissue. I have also annotate the different forms that I'm seeing (pro-LC3, LC3-I, and a dimerized form). If I over-expose the blot I can see a faint LC3-II band, but it was expected for me not to see it strongly in this case. Also, the dimerized LC3 is just a hypothesis of mine, I'm not positive that that is right. For this, I boiled my sample in SDS buffer and electrophoresed on a 12% acrylamide gel, transferred to PVDF membrane, blocked in 4% milk-TBST for 2 hrs at RT, and incubated overnight at 4 deg in a 1:100 dilution, washed 3x10 minutes in blocking buffer, and incubated for 2 hours in secondary antibody (HRP-goat anti-rabbit, 1:5000 in blocking buffer) then washed 2x10 min in TBST, 10 minutes in TBS, and developed with ECL. The journal Autophagy also has a forum with some reviews of antibodies you may want to check out.