loss of protein during concentration
Posted 18 November 2010 - 04:44 AM
i am in a very weird situation. i am trying to concentrate my protein(hemaglutinin+Fab) complex using millipore, 10kd cut off. but my protein concentration reduced to half the total conc. no precipitation is visible. no leakage, as i checked the waste with both bradford and spectrophotometer. i guess, protein got degradede, as there was an extra band in the sds i runned. but the same protein when i complexed with a different Fab, was stable enough. again when i rinsed the centriprep membrane with concentrated protein, i found the concentration to be increased, but not that extent. at this point i am confused whether there is degradation or precipitation, or both. it would be very kind of anyone who suggests me any tip to overcome this problem.
Posted 18 November 2010 - 06:28 AM
In practice it is not that when you use a 10KDa membrane all the protein above that range will stay at the top.It depends on the conformation of the protein, so always don't use a membrane which has a cut off close to your protein's MW. Some proteins precipitate when they are concentrated too much, so determine this concentration first. Maybe the flow through contians the protein and you don't see it because it is very diluted! To prevent aggregation or precipitation we atry to add mercapto-ethanol.And are you doing the concentration at 4 deg?? If not try doing that!
Posted 18 November 2010 - 07:59 AM
Posted 18 November 2010 - 02:21 PM
Do you wash it with the elution buffer...and how much do you add of the buffer to wash it?
Posted 19 November 2010 - 07:22 AM
Using these concentrators there is no binding and elution (as in an affinity column). The concentrators have a membrane with a defined cut-off (which by the way just means that 95% of the protein with that molecular mass is retained) and you wash with whatever you want the end buffer to be. Me, I always use PBS, but you can use whatever. The exact volume depends on the column size. Millipore makes a variety of concentrators and we use the 500 ul, 4,5 ml and 15 ml types. I generally wash with the amount of buffer that completely covers the membrane and just lightly vortex the whole thing a couple of times. If I really want to retain all of the protein I repeat this a couple of times with fresh buffer, but of course this defeats the whole purpose of concentrating.
Posted 19 November 2010 - 12:23 PM
Fab is about 100,000 mw hemagglutinin is about 50,000. A 10,000 mwco should be fine.