loss of protein during concentration - (Nov/18/2010 )
hi, all
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.
thanks.
taslim,
ku, korea
Hi,
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!
cheers
Shiva
In my experience the protein just sticks to the membrane used for concentration. Even though they say that the membrane doesn't adsorb protein it still does. I once read (correct me if I'm wrong) that the membranes are from nitrocellulose. The precipitation or degradation are in my opinion minimal, but just adsorption of the protein to the membrane. Centrifuging at 4 deg. just slows down the entire process. What we do is that we wash the membrane after concentrating and either just add this to the concentrated solution or if this would cause it to be too diluted use this for other types of experiments, where concentration is not such a big issue.
BioMiha on Thu Nov 18 15:59:02 2010 said:
In my experience the protein just sticks to the membrane used for concentration. Even though they say that the membrane doesn't adsorb protein it still does. I once read (correct me if I'm wrong) that the membranes are from nitrocellulose. The precipitation or degradation are in my opinion minimal, but just adsorption of the protein to the membrane. Centrifuging at 4 deg. just slows down the entire process. What we do is that we wash the membrane after concentrating and either just add this to the concentrated solution or if this would cause it to be too diluted use this for other types of experiments, where concentration is not such a big issue.
Do you wash it with the elution buffer...and how much do you add of the buffer to wash it?
sixerguy98 on Thu Nov 18 22:21:43 2010 said:
Do you wash it with the elution buffer...and how much do you add of the buffer to wash it?
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.
Millipore has a toll free phone number. they have great tech support. If they can not answer your question immediately they will call you within 24 hrs.
Fab is about 100,000 mw hemagglutinin is about 50,000. A 10,000 mwco should be fine.
Fab has 50 kDa, F(ab)'2 has 100 kDa!