Problems with my western blotting of LC3, autophagy protein... - (May/21/2009 )
I am a novice in this area, and I am having a great deal of trouble with western
blotting. I wont to detect LC3 protein. I started to use PVDF for western blot recently
to detect LC3. I use 15% acrilamide gels. We have purchased a new and fresh antibody
against LC3 (1/2000). I don't have problems with the electrophoresis (I think..).
Always I dip the membrane on methanol first. My PVDF membrane seems to have only the firsties bands (the heavyest markers proteins) of the marker, and the lightest bands appears faint (my assumption is there was no problem with transfer). I do a block with 5% milk + PBS-T (with 0.3% tween) and primary ab incubation overnight at 4ºC...However, I get poor results: faint bands of LC3 protein (weak signal). Please help¡
Which are the specific conditions to detect LC3? Should I change the strenght of
my buffers or detergents? What should I do?
I would be very glad to receive your recommendations and opininons for my problem.
Any suggestions are welcome!
I remember thet once a student in my lab did a WB to detect the two isoforms of LC3 (if I remember correctly the cleaved form is 16 and 18 KDa...she wanted to see if LC3 was cleaved in her conditions) and she used a Tricine-sodium dodecyl sulfate-polyacrylamide gel to detect low molecular weight...sorry but this was not my work anf I can't tell you more than this!
Hope this helps...have a nice day!
your tween concentration looks a bit high. we normally use 0.1% and, in the literature, i see others routinely use 0.05-0.1%.
you may be following the procedure of someone who had severe background problems which were resolved by increasing the tween and were willing to suffer some loss of signal to clear it up.
the recommendation by velella, to use (tris-)tricine sds-page (shager and von jagow), will help resolve close moving low molecular weight proteins in a lower concentration gel (10% would be okay for the mw that velella cited) than you are using. the lower concentration gel may improve the transfer (if there was a problem).
You could increase your total amount of protein. I use cell signaling LC3 primary antibody(1/500)-it works great. LC3 form 2 is lipidated, not cleaved, and runs faster on the gel than form 1.
Sorry for the delay Vellela¡¡¡But I was working whit yours suggestions and it works¡¡¡Why?? Its a mistery¡¡
Sorry for the delay "d"¡¡¡But I was working whit yours suggestions and it works¡¡¡And, now I have a good antibody that can detect LC3I and II unambiguosly.
Thanks a lot.
I had applied all considerations that you, dtimm and Vellella suggest me.
Now I can get an aceptable gel to LC3. However, I think that to reduce the Twinn concentration to 0,1% was crucial to the success of the procedure. I had read that the PVDF membranes are very sensitive to high levels of detergents, and should never exceed 0,1%.
Thanks for your help.