Hi everyone!
So I am trying to concentrate my recombinant proteins in order to make some assays with them, but I need a "considerable" concentration of each. One of my proteins is around 9kDa and the other one 15.8 kDa. They have been tagged with a 6 His and went already through a Nickel column. SDS PAGEs show considerable concentration of certain amount of protein of interest in many, fragments, hence, it was decided to concentrate them using a PES protein concentrator (3,000 MW concentrators) . However, when spinnning them as protocol suggests and collecting every flowthrough (FT) to localize any "lost" proteins, as well as the concentrated sample, rather than seeing a considerable larger band on the SDS , the bands are discrete, not much concentration is found and there is no protein whatsoever in any other FT to suggest protein is being lost through the filter. Am I facing degradation, or why is it that even concentrating it, is not concentrated.
Thank you all! Welcoming any suggestions or corrections!
Best wishes,
Jorge
The proteins may be sticking to the filter device. Another approach to concentration may be preferable.
One idea is to rebind them to a small nickel column (assuming the imidazole has already been removed by dialysis), and elute them in a single concentrated fraction with a high concentration of imidazole.
Another idea is to concentrate them by freeze-drying. This is best done after removing most of the low-molecular-weight solutes by dialysis, since the solutes will also be concentrated.
A third idea is to place the sample in a low molecular-weight-cutoff dialysis bag and partially dehydrate the sample by immersing the bag in a dry absorbent resin, such as Bio-Gel or Sephadex, replacing the resin occasionally as it swells.
As well as the excellent suggestions from Adam B Shapiro there are a few other things that you could try. Firstly, if you need to use centrifugal concentrators, to reduce sticking to the concentrator membrane you could try pre-treating the membrane before using it. This is very similar to "blocking" like in Western blot, ELISA etc immunodetection with the idea being that sites on the membrane which may non-specifically stick to your protein can be already occupied by another molecule.
There are a variety of agents that you can use for this, with the choice being determined by your downstream application of the protein. Sartorius have an excellent guide to pre-treating concentrators at https://www.vivaproducts.com/downloads/vivaspin-app-notes-3.pdf with suggestions for several different agents (although for their Vivaspin concentrators the same basis applies for other brands and I have used it with several different types). I found this process to be very helpful in preventing losses of a membrane protein that I was purifying and which required multiple steps of concentration during the process.
Of the suggestions from Sartorius, the ones that are probably best for you are (in water) either 5% Triton-X-100 or 5% Tween 20, or, if you want to avoid the possibility of detergent contamination, 5% PEG 3000. I have used all of these at one time or another. I have also used 5% DDM as a membrane protein was being purified into a buffer with a low concentration of DDM and therefore potential contamination from anything else could be avoided.
The process is pretty simple and described in detail in the guide that I linked above. Wash the new concentrator membrane with water by spinning it through and then removing any excess with a pipette. Add your chosen pre-treatment agent (at 5% in water) to fill the concentrator over the membrane and leave it at room temperature for 2 hours or overnight (although Sartorius recommend no longer than 2 hours if using TX100). Next, remove the pre-treatment agent and wash the membrane 3-4x times with water before spinning through. You can now use the concentrator immediately or fill it to the top with water and store at 4 degrees C until needed.
Another suggestion could be to try using absorption style concentrators rather than ones that act on centrifugal filtration (this is similar to one of Adam's suggestions). You can buy so-called "clinical concentrators" that are designed for use with urine or spinal fluid etc samples, but you can use them for other types of sample. This type at https://www.vivaproducts.com/bjp-clinical-concentrators/bjp-20/ hold up to 20 ml with the optional expansion reservoir. The main advantage of these is that they are very gentle and can help avoid loss from sticking etc but offer excellent concentration. These were introduced to me by a college several years ago and although I never needed to use them in the end, other people got excellent results using them. The only real disadvantage is that only a 7.5 kDa cut-off is available, which is almost certainly too close to the size of your smaller 9 kDa protein.
I hope you find these suggestions useful and best of luck!