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semiquantitative western blotting


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#1 natomeru

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Posted 07 January 2005 - 01:35 AM

Hey
We will screen a group of patients who lack or express various amount of a certain protein. One of the techniques used is western blotting and my problem is how do I prove that the expression of this protein is lower in the patients than in normal people.
Others groups normalize the protein with the corresponding myosin heavy chains band on the post-blotted gel (using coomassie blue) but what about the blotting effiency?
Is it more correct to normalise to some other protein (actin eg.) on the same blot stained with ponceau S?
Or is the solution to make a mini-mutiplex western blot with two antibodies, one specific for the protein of interest and the other for a normalisation protein?

Thanks

Mette

#2 ajames

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Posted 12 January 2005 - 09:48 PM

Your last method is the one most commonly used. Actin is probably mostly used although there are other housekeeping genes that people use. The actin antibodies available usually give very high readings so you may want to cut your membrane in half and use the two antibodies on seperate halves so the actin signal does not get saturated-this depends on the size of the protein you are looking at. If the actin band will be too close to the protein of interest you can first probe with one antidoby, strip the membrane and then probe again with actin. It is best to do it in this order because the stripping process will lead to a lower signal and the protein of interest is unlikely to give the very high signal that actin does.




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