how to purify Fc fragment
Posted 30 June 2011 - 10:37 AM
Posted 01 July 2011 - 06:06 AM
Posted 01 July 2011 - 07:37 AM
Posted 01 July 2011 - 08:09 AM
Looking back at what I posted previously...
The IgG capture would retain anything in the sample that had an Fc region. So, I guess if the papain isn't 100% efficient at digesting your Ab then it would bind whatever amount of intact IgG still remains, as well as the Fc fragments.
Have you tried an ion exchange or RP based method of separation?
Edited by proteaMatt, 01 July 2011 - 08:25 AM.
Posted 02 July 2011 - 01:51 AM
Posted 15 July 2011 - 07:07 AM
genius does what it must
i do what i get paid to do
Posted 21 July 2011 - 03:50 AM
Posted 17 August 2011 - 12:43 AM
1) Be sure to work in reducing conditions as disulfide bridges keep the Fc molecules bound together. Therefore, you might have a mix of partially digested dimers (IgG) or pentamers (IgM) after papain digestion.
For that, use 25mM freshly prepared cysteine solubilised in the buffer of interest.
2) As you previously described, Amicon columns with several MWCO do exist in 50mL Falcon format for volume up to 10 mL. If your sample is bigger, you can fill the tube several times. Recovery yield is 70-80%.
3) If you really work on an semi-industrial scale, concentrate your sample in dialysis tube and perform a size exclusion column.