We are working with hcT116 cell line, we transfect the cells with Gapmers and then we want to know whether the genes are bein silenced at a transcriptional and translational level. However, protein isolation and later Western Blot analysis are showing some drawbacks with our proteins of interest, so we'd like to know if substituting WB for immunocytochemistry would be a proper solution for protein quatification. If it is not, what other protocol could be feasible?
Thanks in advance!
Immunocytochemistry will be qualitative and it can localize in cell which part of cell or tissue has the protein of interest, that is important too. But I am afraid, it may not add any accurate data for quantitative measurement.
Hello Paula Vázquez-Fernández
Western blot would have been an ideal method in your case. Also, protein immunoprecipitation would have been good. But you mentioned that WB is showing some drawbacks with your protein of interest.
So I would suggest you go for Mass Spectrometry which is commonly used for identification and quantification of proteins. The protocol involves digesting proteins into peptides, which are then separated, fragmented, ionized and captured by mass spectrometers. It is feasible provided you have a mass spectrometer to work with.
I hope this information is helpful.
Best Wishes.
Dear Malcom Nobre
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