Western blot and isoelectric point
Posted 17 January 2012 - 10:39 PM
Continuing with my previous problem. I found that all my proteins have pI [isoelectric point] more than 9. I read from milipore site that in western blot the pI can affect the protein transfer to membrane.
Methanol strips the SDS from proteins and proteins are finally transferred to membrane - I was wondering whether really the pI will affect the SDS-PAGE followed by western blot OR the pI is imp only if use the proteins in native form ?
Any one faced problems with proteins with high pI [Transfer buffer is pH8.3 and protein pI is more than 9 so proteins will have positive charge and will not migrate towards the membrane]
Posted 19 January 2012 - 09:19 AM
you can try a CAPS buffer at pH 11 for the transfer.
i am attaching a book from millipore and you can download a manual from ge healthcare from this link:
western blotting - priciples and methods
talent does what it can
genius does what it must
i used to do what i got paid to do