The protein purified by them is showing binding to the RNA. I have used exactly same lysis, washing and elution buffer. I have got purified very good purified protein.
Hi there,
The most obvious reason would be that the protein is not properly folded or unstable during the assay.
Does your protein has compatibility to RNA? The extraction protocol is not much of a relevance if there is no compatibility between the protein and RNA. Do you have same protein and RNA as mentioned in the reference paper? Do you try to establish a new relationship between protein and RNA of your interest or it is a confirm study that your protein will bind to RNA?
Hello,
maybe your protein is very good purified, but is not "active". For example, in case of elution buffer contains imidazole, the subsequent dialysis step on commercial columns (https://www.thermofisher.com/cz/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration.html) is necessary to achieve specific protein binding.
Hope this help.
Thank you for your response. Yes my protein is previously known to bind to this RNA. My protein is gst tagged. The buffers used for lysis contain 50mM tris, 500mM NaCl, 1% triton x, 30% glycerol, 1mM DTT, 1mM EDTA and binding buffer contains 50mM tris, 200mM NaCl, 1% triton x. The only difference between the paper protocol and my protocol is they have lysed the cells using lysozyme while i have sonicated cells. Will this affect the protein?
Hi again,
Lysis method may have an impact on protein integrity. Sonication being much more violent than osmotic shock on spheroplasts.
Hi debasish,
apart from mentioned reasons above,just check weather your protein of interest has different isoforms?if so then check which isoform has RNA binding activity.Sometimes it happened that RNA binding ability is isoform specific.Also u can think of alternative methods to show RNA-protein interactions such as Biotin pulldown and RIP.
Does your purified protein contains still nucleic acids? maybe this can be the problem of not binding to your RNA. Try to use ion exchange chromatography.
Good luck.
Hello Martin Bartas,
Why do yo think my protein would'nt be active? What can affect its functionality? Can anybody please suggest me how can i obtain active form of protein?
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