I tried the Abcam antibody in western blotting, but when I reveal the membrane appear some non specific bands. Even though I left the membrane blocking for 2 hours in 3% albumin solution.
If you see band of your protein size plus other bands then dilute the antibody (primary or secondary) more or wash them after incubation for several more time to get rid-off the background.
Thank you Muthusamy for answer my question. I have diluted my primary antibody 1:750 instead of 1:500. But I will try wash one, maybe two times more when I add the secondary as you suggested. It helps if I wash once more but after adding the secondary too, before reveal it?
I would add primary 1:1000 and wash after primary 3 times 5 min and then incubate with secondary as suggested by the company (usually 1:5000 to 1:10000 or more depends on the company). then wash 3 X 10 min and develop with low sensitive kit to high sensitive kit (clarity---> Dura----> Femto).
But I fear that if I dilute more my primary antibody, I do not get to see the band that I need to see, because of how I've been doing it almost does not appear, as you can see in the picture where I circled in red
this helps. Could you tell me what is the secondary ab dilution? what is the kit you are using to develop the blot?
The secondary ab dilution is 1:30.000. We don't use kit, we use the reagents made "in house".
Dear Muthusamy, I'm trying incubate with a 5% albumin solution to blocking inespecific ligation for 2 hours. I will post the result here. Thank you for your help anyway.
Hello,
I would recommend an antibody database available at www.labome.com. For antibodies to HGF, you can check this link: https://www.labome.com/review/gene/human/hepatocyte-growth-factor-antibody.html. In addition to mentioned Abcam antibodies, Santa Cruz Biotechnology has rabbit polyclonal (H-170) HGF antibody (sc-13087) was used in western blot on rat samples (reference: Yi et al. Int J Mol Med 2014). I hope this comment is helpful.
- Badges
- Science method