Hi all,
I noticed small white precipitates formed during buffer exchange using Vivaspin. I am new to protein biology. I am not sure if the white stuff that I see are protein precipitates or something else from the column? salt? Any suggestions please? I would also like to know once the protein has precipitated can it go back to the solution if mixed thoroughly?
Thanks,
Kiran.
What may be happening is that during the concentration in the Vivaspin, the protein concentration exceeds its solubility in part of the concentrator (against the membrane or at the bottom) because it is not well-mixed. In the rest of the concentrator, the protein concentration is lower. When you mix everything after the concentration, the overall protein concentration is lower, allowing the precipitated protein to dissolve again. This is not a case of protein denaturation, otherwise the protein would not redissolve. There are 2 ways to deal with this: avoid overconcentrating the protein, or identify buffer conditions in which it is more soluble (change the pH and salt concentration).
The visible white precipitates are most likely your protein aggregates.
Probably your protein is not very happy in the buffer and is hence crashing out of solution. What you need to do is optimize your buffer conditions and see what keeps your protein (and you) happy. Also, see if too much of concentration is causing your protein to start precipitating. Play around and optimize.
To your last question: Unfortunately no, once your protein crashes out of solution mere ''mixing thoroughly'' will be of little help. One approach could be to denature your whole protein sample in 6M Guanidium hydrochloride buffer and then attempt to refold it in vitro. But that is a whole different ball game needing a different skill set. Try that only if everything else fails. Feel free to ask for further details if necessary.
Best of luck.
Hi Aditya,
Thank you very much. This is my second time extracting and purifying the same protein. I dint have this problem in the first instance :( but anyways I will tray and make changes to the buffer.
The weird part is that the white precipitates goes back into the solution upon mixing. Does that mean the white precipitates that I see are something else other than protein precipitates?
What may be happening is that during the concentration in the Vivaspin, the protein concentration exceeds its solubility in part of the concentrator (against the membrane or at the bottom) because it is not well-mixed. In the rest of the concentrator, the protein concentration is lower. When you mix everything after the concentration, the overall protein concentration is lower, allowing the precipitated protein to dissolve again. This is not a case of protein denaturation, otherwise the protein would not redissolve. There are 2 ways to deal with this: avoid overconcentrating the protein, or identify buffer conditions in which it is more soluble (change the pH and salt concentration).
Great. If upon mixing the precipitates are going back into solution then it is because there is local change in your protein concentration while you are concentrating it. Try mixing up the solution at intervals while concentrating. And be careful not to make the concentration too high.
You might want to consider another option to look at this problem. Some salts after mixing might generate some precipitates and form insoluble particles as a product of chemical reaction between them. You need to make sure that all components of your two buffers are not producing any insoluble salts after mixing during the buffer exchange.
Thank you very much Adam and Aditya. I spun down the protein tube today and found quite a bit of the white stuff. I mixed them up pipetting up and down. Almost everything dissolved! I performed Bradford assay to determine the protein concentration. There is no colour change!!! At 595nm both my blank and sample have almost the same reading! The protein was aliquoted in 100 ul fractions in eppendorf tubes. I mixed my protein solution in each tube thoroughly before putting them all into one tube for centrifugation. I added the Bradford dye directly into one of my empty tubes with protein and it turned very light blue. What does this mean? the white stuff were actually protein precipitates? When I performed the Bradford assay immediately after protein purification (the white stuff was present at this time) there was 525g/ml of protein.
Hi Viktor,
Thanks for your reply. I will look into it. But my concern now is what happened to my protein?
Hi,
Using concentrators for buffer exchange is not the best as Adam has already pointed out-your protein may be soluble in your buffer at a low concentration but not at higher concentrations.
To avoid concentration I would use either dialysis or, even better, a desalting column. There are many formats available for just about any volume you would need to process for manual (spin or PD-10 gravity) or prepacked columns for FPLC systems.
- Badges
- Science method