purification of a basic protein
Posted 10 March 2009 - 08:09 AM
Posted 10 March 2009 - 05:52 PM
Posted 11 March 2009 - 07:10 AM
Posted 12 March 2009 - 03:50 AM
I also face this problem of protein not binding to beads under denatured conditions. I refold the whole lysate and then bind.
Alternatively, give a lot of washes with 1M urea and get rid of the proteins soluble in 1M urea. Now give repeated 2M washes and run the fractions on a SDS-PAGE gel. Refold the ones which are relatively pure, bind it to an anion exchange column and purify or change the buffer.
Posted 13 March 2009 - 09:12 AM
In reply to paramyosin - using such strong denaturants like 8M urea or guanidine might denature the GST or column to an extent that it might even affect binding. maybe using 8M urea for just the elution can work.also i was wondering that since the binding of the GST tagged protein to the glutathione column is based on a covalent disulfide linkage would it worth including a reducing agent like mercaptoethanol in the elution buffer. any suggestions?