I am doing RNA-Protein interaction exp. I want to see binding by filter binding assay, I used Millipore PVDF 96-well filter plates, with vacuum manifold, but could not see binding, I also tried with Millipore MCE membranes, but the results were same. RNA is radioactive labelled. Even in control there is some binding but in tests it is not more than control. My protein is in 40% glycerol apart from usual buffer, it has an RNA binding motif so it should must bind. EMSA showed binding.
Hi Rakesh,
For PVDF membranes to bind proteins, they need to be pre-activated with 100% Methanol prior to use for transfer. Have you done this?
If you did and still don't see any better signal to noise ratio relative to control, maybe you need to work on the cross-linking (UV-C 254nm or 4Thio-Uridine+UV-A 365nm).
Hope this helps,
Utkarsh
Do you have a good positive control to establish your filter assay?
In case you need an independent method to verify EMSA results, I would suggest MST (MicroScale Thermophoresis). It's a fast, easy and precise method to quantify biomolecular interactions, thus you would also obtain binding affinities for your RNA-protein interaction. You could also easily quantify binding of different RNA substrates or protein mutants.
I attached a paper discribing MST and showing some interesting examples.
Agree with Utkarsh above. Many experiments fail because the PVDF membrane is not activated before usage..For example, during dot-blotting or Electro-transfer of proteins, if the PVDF is not activated (after rinsing with Methanol,it will turn from white to a greyish color), you will not be able to see any protein transfer afterwards..
Best
Hediye.
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