I isolated my protein complex from mammalian cells. Purify it using affinity purification methods. but after running chromatography the concentration became very low to go further structural study. How can I I increase concentration without affecting the protein complex?
@Omar Gonzalez-OrtegaL Any other option? I tried the amicon filters but not so satisfied.
Why are you not satisfied with Ultrafiltration? Are you sure you are using it correctly?If your volume is too big and your protein is too diluted, it is the best option.
Make sure you pre-activate your membrane before you use it. Also, there is a direction to the membrane. Be sure to use the side that is "glowing" when you put it in the light. If you do not use it correctly, you will lose all of your proteins. You can check this by adding 10-20 ul into Bradford solution. You shouldn't be losing much.
If I were you, I would use amicon filters. If the concentration is not desired, I would use protein concentrator spin (small one). At the end, I would spin down and get the soluble fraction.. Why don't you just use amicon filters? I feel like it's your best bet. If your volume is relatively small, you can use Vivaspin concentration, but this takes time.
Lastly, you can precipitate your proteins using NH4Cl and reconstitute it. Of course, GPC or GF to replace the buffer. Dialysis is not a way to concentrate protein.
If your volume allows, I would suggest using Centriprep filters instead of other filter tubes. The Centriprep tube's mechanic is the opposite from other tubes, such that the protein sample is underneath the membrane and the centrifugal force will push the liquid through the membrane from below. That way you don't run the risk of losing your protein due to binding to the membrane. Funny that in principle, proteins should not stick to these membranes, but it's harder to get all of your protein from the membrane than from the bottom of the plastic tube. This is quite clear when your protein has a cofactor that is colorful. We have made a lot of recombinant proteins and whenever we use regular filter tubes with the protein sample above the membrane, we lost a significant amount compared to the Centriprep tubes.
Just my 2 cents. Good luck!
I would suggest dialysis in a 3-6 kDa MWCO membrane against 40% PEG20000 in the same buffer of proteins
If the volume is large enough then ultrafiltration works well. A difficulty can arise on occasions if the quantity of protein is low. Then the protein may stick to some types of membrane and be lost.
A simple method is to place the protein solution in a dialysis bag and pack sucrose around the bag and place in fridge. The moisture will wick away from the bag into the sucrose concentrating the solution of protein after about 2 hours. The sucrose can be replaced for further concentration.
The final protein solution will need subsequent dialysis (in a smaller volume bag) against a suitable buffer to remove sucrose if it is a problem.
Learn something about dialysis for concentration @Woojong Lee from the previous answers
I'm assuming that your protein complex is fairly stable. If so, you could try a very gentle ion exchange. If you use a small column and optimise the elution well, then often the output is rather more concentrated than the input. As your question tags suggest that you are aiming at cryo-EM, this should allow you to get the protein to the sort of concentration that you need - it wouldn't be enough for crystallography!
Another approach would be to repeat your affinity step if you can (use a small column and a step elution). The output from that should be far more concentrated than the GF output.
Hi Abhijit, I agree with Nicholas that repeating your affinity purification step is probably the easiest way to concentrate your protein complex - unless you used protein tags for the affi purification that were then removed during subsequent purification steps.
Thank you @Nicholas J Harmer and @Steingrimur Stefansson I will follow your instruction as well. Yes, my protein contains FLAG tag and I am doing affinity purification. I am trying to isolate protein complex. The purification is ok. but the concentration became lower every time.
Can you suggest any specific protocol or article follow your guideline? my email id: [email protected]
Please send me.
@Woojong Lee I already tried Ultrafiltration using amicon filters according to the guideline. Thank you, I will try it again with more concern.
Use Vivaspin filters to concentrate, they are good to concentrate (my personal experience), and you will not loose much protein when compared to other filters. You can have different size MWCOs.
- Badges
- Science method