AnionExchange problem: proteins do not bind - (May/27/2010 )

sorry for responding so late,
The column does bind some protein (only a little in comparison with the flow through peak (~aprox1%). If I scout for another protein via WB it seems nicely bound, but my protein of interest isn´t. The problem is, that it was retained once (my initial experiment) but ever since my protein of interest doesn´t bind any more...
Unfortunately I can´t measure conductivity...

Different proteins bind differently in a resin!, but if you have good binding of other protein is because the resin is ok. Maybe your initial conditions were different, more NaCl concentration?, different pH?, different resin-protein relation?

hi fren...

by the way u have to see the principle of Ion exchange chromatography...let me explain..

since u use Q resin....i do believe it is positively change resin.if u want to make ur protein bind to the resin, u should make ur protein as -vely charged.HOW?????

Do u know the isoelectric point of your protein????if not, u should make an isoelectric focussing (IEF) experiment...then, from the pI value that u will get, u can know what is the appropriate pH for ur sample to ensure the it will bind...

i give u example, let say the sample is IgG....u spin down ur sample and run IEF in order to get the pI value...
let say the pI is between 7.6 to 8.2....by assuming an electrophoretic titration curve, i.e. lower the value from 7.6 will make the protein as +vely charge, whereas increase the value from 8.2 will make the protein as -vely charge...

let say u want the IgG bind to Q resin, so u must choose pH above 8.2...let say u set pH at 8.6

Then, u choose any buffer that has buffering region at this chosen pH....at pH 8.6, Buffer Tris-HCl (10 mM) can help...then, u dialyze/ buffer exchange ur sample to this buffer...

Secondly, by rule of thumb....for ion exchange chromatography binding mode (i.e. product of interest will bind to resin), the conductivity of sample must less than 5 mS/cm...so, use buffer at low strength...such as 10mM or 20 mM....


by the way, good luck...