Hi Everyone,
May i ask who has normalized his/her IP results for interaction? especially considering equal over-expression, equal loading into a gel and equal pull-down across samples?
Thanks,
Hey, I'm not sure if I can be helpful in this question, but could you shortly explain why it is needed in your experiment to normalize your IP results?
Thanks in advance!
Hi,
Normalisation of IP isn't the same as normalisation of a WB (in which you can use actin as a loading control).
You normalise your band intensity to your control due to the fact that you used the same amount of protein in incubation with the same quantity of antibody and also that you load the same volume of pull-down from each condition.
That's all I can say about the normalisation of IP.
Good luck
Stephane
Thanks Ariane and Stephane for your replies. I did many mutations on a protein, separately. Therefore, when I overexpress those proteins, they do not express equally, resulting in a different amount of pulldowned protein and interaction. So, the problem happens in the beginning and I tried different ways to handle it like MG132 treatment, in order to prevent my mutant proteins' degradation. However, it did not work for all. Hence, I thought that normalization containing normalization of expression level, pulldown etc. would be better. (Btw, I transfect those mutant proteins with another wild type protein (so, co-transfection))
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