Protein concentration problem ! - (Jun/05/2007 )
Hello, 
I'm concentrating a protein which comes from Sigma. There are 5 tubes of 0,5mg/ml, I resuspend each tubes in 1 ml of buffer pH 6,5 (pi of protein 7,2), finally i have 5 ml of solution at 2,5mg/ml. I concentrate with centricon of santorius, with the good MWCO, but I lose proteins, I have a concentration of 0,6mg/ml in 1ml of my solution after concentration by centrifugation.
Welcome any kind of discussion and idea. Thanks.
I'm concentrating a protein which comes from Sigma. There are 5 tubes of 0,5mg/ml, I resuspend each tubes in 1 ml of buffer pH 6,5 (pi of protein 7,2), finally i have 5 ml of solution at 2,5mg/ml. I concentrate with centricon of santorius, with the good MWCO, but I lose proteins, I have a concentration of 0,6mg/ml in 1ml of my solution after concentration by centrifugation.
Welcome any kind of discussion and idea. Thanks.
protein loss by unspecific binding of plastic and filter membrane is known but your loss is dramatic! did you determine protein conc before centrifugation or did you only calculate? may be there was less protein in the vials as declared...
I'm concentrating a protein which comes from Sigma. There are 5 tubes of 0,5mg/ml, I resuspend each tubes in 1 ml of buffer pH 6,5 (pi of protein 7,2), finally i have 5 ml of solution at 2,5mg/ml. I concentrate with centricon of santorius, with the good MWCO, but I lose proteins, I have a concentration of 0,6mg/ml in 1ml of my solution after concentration by centrifugation.
Welcome any kind of discussion and idea. Thanks.
I don't understand. The original tube is 0.5mg/ml. You have to resuspend, so the provided protein was in lyophillized form? The original concentration is 0.5mg/ml, but the total protein in each tube they sold to you is???? It you resuspend in 1ml and get 2.5mg/ml then does it mean each tube originally got 2.5mg? Are you sure of this number or is this each tube at the beginning only got 0.5mg? If so, when you resuspend in total 5 ml (for 5 tubes), the concentration should still be 0.5mg/ml.
Still, even with starting 5ml of 0.5mg/ml, after concentration, you get 1ml of 0.6mg/ml, right? Then it is still lost too much protein. You sure the centrifuge column is within the suitable range of protein size for your protein? If the column is for, say minimum 30kD protein, and your protein is 20kD, then you will lose it. Did you discard the flowthrough, or do you still keep it? If you still keep it, can you check if your protein is in the flowthrough?
The protein from Sigma is in lyophillized form (indication on the tube is 0.5mg/ml on each tubes). When i resuspend each tubes with 1 ml of buffer and i poll them, then i have theoretically 5ml at 2.5mg/ml. Size of my protein is 30Kda, and i use centrifuge column with MWCO 5000, i don't use centricon with MWCO 10000 because i don't have them.
I check the flowthrough and there is no protein
You have 5 ml with concentration not 2, 5mg \ml but 5 ml with concentration 0,5mg\ml
Only if you' ve used 1 ml to dissolve powder in all 5 tubes only so you achieve 1 ml with conc 2,5mg\ml well?
Are you sure ??
Are you sure ??
yes
Excuse me, i made a mistake. on tubes it's indicated 0.5mg. and in preparations instructions it's indicated : "the product is soluble in water (0.5mg/ml), yielding a clear to slightly hazy solution"
If you put 1ml in each tube, then you will end up with 5ml pooled at 0.5mg/ml.
Protein doesn't just disappear, so it must be somewhere. Either stuck to the centrifugation apparatus or it is in the flow-thru significatly diluted. So, you need to concentrate the flow-thru in order for you to measure it (most likely).
In the future, you could put less than 1ml into each tube to get a more concentrated result. For example, only put in 0.5ml and you would have 1mg/ml instead of 0.5mg/ml.
You will always lose protein when you concentrate any sample, but you should not have lost that much. Did you look to make sure that there was no lyophilized antibody still on the inside of the tube's top? Sometimes the lyophilized particles get everywhere inside the vial and not just settled on the bottom.
it says that the protein is only soluble to 0.5 mg/ml. you tried to concentrate it to ~2.5 mg/ml. it probably precipitated and is coating the membrane (unless you rinsed it, then it is gone).
did you really need to make it more concentrated than they recommended?