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I ordered the anti-TIM1 antibody Ab47635 but I have been having no success with western blots. I am trying to detect KIM-1 (TIM-1) in lysate from whole kidneys. According to many published papers, KIM-1 increases in the kidney following injury. However, when I run my samples on a western blot 1) there is no difference between non-injured kidneys and kidneys from animals with a significant rise in serum creatinine and 2) in both samples I am getting upwards of 15 bands at all different sizes. I have tried blocking with both 5% Milk and 5% BSA with no difference in the results. I am currently working on confirming that KIM-1 mRNA is increased in the injured sample by qPCR. I am using a standard NP-40 based lysis buffer. Any suggestions?
Asked on Feb 22 2012
Thank you for contacting abcam.
The image on our datasheet for this product ddoes show high non-specific background as well which seemed to be tamed but not eliminated by using a much lower dilution of the product. In that particular case 1ug/ml versus 2ug/ml with 15ug of total protein per well. Althought the exact concentrations will be different with each protocol and tissue type the principle should hold. I would recommend that you titer the ideal antibody concentration at a much lower level.
Another recommendation is to use a BSA block before and with your primary antibody but to use a casein based commercial block for incubating your secondary antibody. We have seen that this may dramatically reduce background in western blot. We do carry this type of product:
Background Reducing Buffer (https://www.abcam.com/Background-Reducing-Buffer-ab64234.html)
Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.
We do offer a
Answered on Feb 23 2012