I have a problem and am asking for advice.I am doing WB. For electrophoresis
I use an 8% separating gel and a 4% thickening gel. The electrophoresis has 2 phases : 30 min-90V , 60 min 110V. Electrophoresis buffer from Bio-Rad of composition 10xTris/Glycine/SDS. For transfer I use Bio-Rad's ready-made but diluted Transfer Buffer. I perform a standard transfer to PVDF membranes (30 min). Membranes are incubated with milk and with tris pH=7.6 and tween. Then as primary antibody I use B act polyclonal antibody from Invitrogen, Lot YD371542, as secondary antibody anti-rabbit IgG HRP Conjugated HAF 008 from R&D, Lot FIN 1922041. Then I use Precision Protein StrepTactin-HRP Conjugated 5,000x. And I add calling reagents.
Why does my 70 kDA stain very clearly and my beta actin stain very weakly ( 42-46 kDa) ?
I make WB from homogenised cardiac tissue. To prepare it, I used Thermo Scientific protease inhibitor at 225 microlitres per 25 ml homogenization buffer.
I attach a blot of beta actin below .
Thanks for including the blot pic. Your electrophoresis looks very nice. I suspect the StrepTactin reagent is cross-reacting with something 70k in your samples. To investigate this possibility you might try leaving out that step/reagent, or cutting the MW marker band off of your blot and developing it separately.
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