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LC3 antibody advice


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4 replies to this topic

#1 LC3

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Posted 12 January 2011 - 02:30 AM

I would like to do a LC3 immunoblot on human monocyte derived macrophages. Could someone recommend an antibody manufacturer.
Should i pick poly/mono-clonal? LC3 or the more specific LC3-B? are there any protocols available?
thanks

#2 knuf

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Posted 12 January 2011 - 07:08 AM

I have used the LC3 antibody from sigma with sucess on both IHC-P and western blot. This particular antibody is specifically designed for IHC-P on human tissue, though (and is quite expensive) and may be more than necessary for a simple western blot.

#3 antibodymania

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Posted 12 January 2011 - 12:55 PM

Here's a list of LC3 antibodies filtered for Western Blots

http://bit.ly/fxXmKZ

:)

#4 LC3

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Posted 13 January 2011 - 06:25 AM

did you do a WB on human macrophages? would you mind posting a picture of your WB and sharing your protocol?

#5 knuf

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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.

Attached Thumbnails

  • LC3 Western Blot.jpg





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