I try to do Western blot for LC3 band, but i can only see just one band.
I do 16% running gel 90V 6h, 25V 1.5h to transfer, and i use primary antibody - Cell Signaling antibody #4108, then HRP conjugated anti-rabbit antibody IgG, followed by ECL.
I use NC membrane, pore size 0.2um. But i can see only one band.
What do i change conditions? I need your helps.
Quite simple: according to article "LC3 and autophagy" (Article LC3 and Autophagy
) , LC3 shows two similar forms: LC3-I and LC3-II. While LC3-I is quite common, LC3-II occurs with autophagosomes only. Therefore LC3 can be used to determine the presence (or absence) of autophagy. One band (about 17 kDa) should be always present and additional band (at ~14 kDa) will indicate the presence of autophagosomes. If you have only one band at 17kDa, it means that most probably you don't have the autophagosomes in your sample.
First, try any drug that induces autophagosome formation (positive control) and see if you can see two bands, so you make sure that the problem is not with your samples or treatment. Also I used to get two bands LC3 I and II, after running my samples on 12% gel, try that out and see if this works. Good luck!
It is important to put the positive control in any experiment including WB analysis of LC-3. Firstly, treat cells with bafilomycin A1 or chloroquine which disrupt the autophagy-lysosome flux and increases the LC3-II accumulation level.
Also simple starvation (removal or reduction of FBS in culturing medium) may also do the work, if you cannot afford compounds mentioned above. As far as I remember, 12-24h of such conditions were good enough to induce detectable autophagy in my collegue's breast cancer cell culture.
Thank you for your answers. First, i will use positive control (without FBS sample) for western blot. And then, i will use inhibitor with material sample. Thank you.
Besides has anyone detected LC3 in NC membrane?
And why do people use PVDF than NC membrane for LC3?
I saw artilce, most of all (but i think all of them..), they used PVDF.
I think that membrane is so important, but pore size is important too.
Its quite trivial question; the comparison can be found here [https://bitesizebio.com/20807/insane-in-the-membrane-pvdf-vs-nitrocellulose-which-one-comes-out-on-top/] and in many other places. Just search for "Nitrocellulose vs PVDF".
Go J Yoshida thanks for your answer to the relevant person. It seems that you are regarding Bafilomycin A as an inducer?
According to my knowledge, Rapamycin is an inducer and Bafilomycin A is still debatable .. since some papers say it inhibitor while some postulate it as an inducer. Correct me if I am wrong.
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