Our group is trying to pull down AGO-FLAG protein to look for miRNAs interacting with AGO2, however we are getting expression at the same Ct for non-specific miRNAs in the negative control group as well in the real time. How to improvise our technique to get good results ?
C K Gomathy
Fixation. Over-fixation can cause high levels of background in IHC experiments. ... Slide Preparation. Decreasing the thickness of tissue sections can help keep reduce background staining. ... Deparaffinization. ... Blocking. ... Antibodies. ... Endogenous Peroxidases and Biotin. ... Further reading: Adjusting the pH of your buffer. Using protein blocking additives. Adding non-ionic surfactants. Increasing salt concentration.
hi,
Non-Specific binding (NSB) refers to an occurrence of an antibody binding to unintended proteins, receptors, or transporters. This binding of assay antibodies is not correlated with the specificity of the antibodies.
ips for Reducing Non-specific Staining in IHC
Strategies to reduce non-specific binding
For more information kindly refer this link:
https://www.abcam.com/protocols/immunoprecipitation-troubleshooting-tips
Best wishes..
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