Dear colleagues. I'm trying to perform a WB to validate antibodies and I'm using Human ApoB100 (>95% SDS-PAGE) as a standard in the gel. I have tried two different loadings of protein 1.5 and 4 ug (boiled 10 min 95-100 ºC in presence of DTT and NuPAGE LDS 4x). Gel runs fine apparently, and after the transfer step to nitrocellulose membrane (2h cool room, 350 mA) I stain the membrane with Ponceau S and I can not see anything there, neither with the coomassie staining of the gel. I used to run the gel at 100 v for 1h 15 min, stopping it just when Bromophenol Blue is already out of the gel.
First WBs were loaded at 1.5 ug of ApoB100 and of course, I couldn't see any band with Ponceau. However, after WB assay (with all the corresponding steps like blocking with non-fat dry milk at 5% in PBST, etc.) I saw like a smeared band (see picture, band at the right). Antibody used in this experiment is a polyclonal antibody against the whole molecule at 1:2000 and secondary antibody-HRP labelled at 1:6000
Does anybody has experience with ApoB in WB analysis? Any advice will be very welcome!
Thanks!
Dear Alejandro Hernández-Albors,
You can follow my article "Effect of urotensin II on apolipoprotein B 100 and apolipoprotein A-I expression in HepG2 cell line ".
Good Luck
Thank you for your comment, Amirhosein. I will read carefully your paper. Probably the problem is the amount of protein loaded. I will try to load less protein, for example 1, 0.5 even 0.01 ug.
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