Hi all , I have isolated proteins from Mesenchymal stem cells directly by using 3X loading buffer. I have done amido black staining for quantification. Further I have also done normalization by using GAPDH so that I can load equally. But in both the case I am not able to load equal protein in all the lanes. Can I use ponceau stain for normalization and quantifying the protein by Image J.
If I understood you correctly you are trying to load the same amount of protein on your PAGE by comparing the intensity of the loading control GAPDH or of a whole protein stain (which can indeed be used for normalization). This is actually not possible as you have no information about the linearity, e.g. sample 1 shows two times the signal of sample 2. In this case you can not simply assume that you have to load the double amount of sample 2 to achive an equal loading.
I would reccomend you to make an isolation with an lysis buffer like RIPA. Afterwards you could do a protein quantification compared to a standard curve. This will allow you to load a specific amount of protein. If you take the matrix effect of your loading buffer into account when preparing the standard curve for quantification it propably also works with the samples you have already. But I never tried that.
Some people even reccomend using a whole protein stain instead of just two or three loading controls. In this case I would reccomend using a fluorescent dye (e.g. SYPRO Ruby) that can be detected on the membrane in a suitable imaging system. Then you overcome the problem of scanning the membrane that makes a densitometric quantification always a bit problematic.
Best regards,
Daniel
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