Hello, I have been attempting to purify a recombinant protein fused at the C-terminus with Twin-StrepTag. For that I am using the Strep-Tactin XT 4Flow High Capacity Spin Column Kit by IBA (Catalogue number 2-5151-000 for reference).
The main goal of this purification is to identify the binding proteins of this recombinant protein.
After following the manufacturer's instructions, I have been unable to detect my recombinant protein in neither the washing nor the elution fractions I obtained.
*Few key points*
- I am transfecting HEK cells with my proteins (both the recombinant and its binding partners) then lysing them
- It was suggested to decrese the volume of the high capacity resin suspension from 100 uL (corresponding to a binding capacity of 600 ug of proteins) to better match the amount of proteins I have. So I pipetted 20 uL of the resin suspension (corresponding to 120 ug of proteins) and the problem persisted
- As a positive control I was able to detect the recombinant protein and the binding proteins in the lysis sample
~~~
Does anyone have any suggestions on ways to elute my tagged protein? Also, If anyone was successful in using this kit can you please provide me with your protocol?
Many thanks in advance!
It can be difficult to elute off Streptactin resin using the manufacturer recommended protocol. When purifying membrane proteins I've frequently found that it is neccessary to use multiple additions of the 50 mM biotin elution buffer and leaving the elution buffer to incubate with the resin for 15 minutes or so can also help.
If you just need to identify the proteins, rather than obtaining them as purified protein, you could try eluting with SDS-PAGE loading buffer and then loading directly on an SDS-PAGE (heating the resin with the buffer may help also, but the denaturing effect of the SDS in the loading buffer will be enough for loading on a gel). If you have access to mass spec, you can also do an on resin digest without having to elute the proteins.
Did you check whether your protein still can be detected in the flow-through?
Leonard Sebastian Fresenborg
Yes, I checked and couldn't detect my tagged protein in the flow through. It's worth noting that I've checked both the washing and the elution fractions and neither showed any signal (just to exclude the possibility that somehow my protein was lost in the washing steps).
Samuel Davis
Regarding your first point, the company supplied elution buffer is indeed 50 mM, however their recommended incubation time is 5 minutes only. I will look into increasing this incubation time and hope there is some eluate.
Secondly, that is a very interesting idea. Didn't consider that the resin could be eluted using strictly SDS-PAGE loading buffer. Will definitely look further into this in case point #1 didn't work.
Finally, yeah this is probably going to be the last resort in case I couldn't elute my proteins.
Thanks for the suggestions!
Ali El Bizri try adding the elution buffer, wait about 15 minutes and collect the elute, then add some more more elution buffer, wait again and elute, then repeat a couple of times. You'll probably find that you'll start to get increasing levels of your protein eluting with each addition of the buffer. For the membrane protein I was previously working with, it took about 5 additions of my elution buffer (containing 50 mM of d-Biotin, but slightly different from the IBA version to suit my protein) to get maximal elution - maximal levels started to come off with the second or third addition of elution buffer. It did help that my protein was mEmerald tagged, so I could visually see how much had likely eluted based on how green the resin remained. I've attached an example from my PhD thesis, ~3 ug of protein was loaded per lane.
I like to use denaturing elution off resin to confirm if a protein was actually bound and present. For larger scale purifications, before the elution step with the normal elution buffer, I just take a 10 or 20 ul sample of the bound resin and add it to 1x SDS-PAGE buffer then heat gently. This can then be directly loaded on a gel, although a brief spin to collect the agarose resin at the bottom of the tube is a good idea. When run alongside the rest of the samples on a gel, it can give a good idea of whether the protein was actually there. Adding resin and then eluting with loading buffer can also be a good way to concentrate samples prior to SDS-PAGE if the level is too low for detection by blotting etc.
Samuel Davis Will definitely try this longer incubation. Also, elution using SDS PAGE buffer to concentrate the sample sounds appropriate for my particular experiment.
Many thanks!
Hey Ali El Bizri,
Could you solve the elution problem? Let me know in case you still have trouble with your protein purification. I'm happy to help!
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