Immunoblot with wrong band weights

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megankmtu
megankmtu's picture
Immunoblot with wrong band weights

I have been performing western blots to detect IL1a from porcine tissue samples. I've extracted my protein using non-denaturing lysis buffer. Before loading in a gel, I boil my samples with 10% B-mercaptoethanol for 5 minutes (100C). I've used a protein standard (recombinant porcine IL1a) and the bands for the recombinant protein show up where they are supposed to (17kDa). However, the bands that appear from my samples are not present for all samples, and appear at ~65-68kDa. I am getting one band at a lower molecular weight from the lane that presents the strongest 68kDa band.

What is going on? Why is my protein so high? I've used both monoclonal and polyclonal antibodies, both with similar results.

varsha
varsha's picture
I have three suggestions.

I have three suggestions.
1. Use a denaturing lysis buffer (e.g. SDS containing Laemmli buffer). That would show resolve the size of monomeric IL-1.
It is possible that you are looking at a complex between IL-1 and another protein/ receptor which is not dissociated by Beta ME.

2. The 65-68 kDa band is another protein (unrelated to or shares a partial epitope) which is recognized by your antibody. You should look at the sequence of IL-1 fragment used to generate antibody. Also try the pre-immune serum in blot to see if it detects the 65kDa band.
If you have a pig cell line or knockout which does not make IL-1B, that would be good control for the 65 kDa banc.

3. You are looking at the IL-1 precursor (31 kDa) which can undergo glycosylation and phosphorylation and possibly other post-translational modification. use a cell line (human or pig) under stimulating condition as control.

Although I have never done an IL-1 blot, I think I would do denaturing PAGE and blot followed by other 2.

Good luck with your blot!

qiang wang
qiang wang's picture
it may also be protein

it may also be protein aggregates, use 4-6M urea in gel