I'm working wiht RAD51-6HIS using Ni-NTA column i'v got the following chromatogram (attachement). There are almost no peaks. I haven't done SDS page yet but, I want to do Ni-NTA one more time first. What could I do to get better results?
200 mM NaCl, 30 mM imidazole -wash 200 mM NaCl, 250 mM imidazole - elution
Lysis: 3M NaCl, Tris-HCl pH 7.5, 4 mM EDTA, 1 mM DTT, protease inhibitor, 100 ml
Thanks.
Did you check the pellet after centrifuging the lysed cells to see if the protein was there?
Also, is there any particular reason why your lysis buffer contains 3M NaCl? That seems extremely high and is actually high enough to denature some proteins.
Check whether the lysis was OK or not and if your protein pelleted or is still soluble. 3M of NaCl is very high, i think that some columns are compatible to up to 2 M. 4 mM of EDTA is also high to be passed into the column, try putting less.
And do the SDS PAGE to check whether your protein is in the Flow Through or not.
Hi, Adam, thank you for the answer!
No, I didn't check the pellet after centrifuging. The time we have at the lab is really limited.
I've got this protocol from the institute I'm studying in. So my first question was also "why do we have such a high concentration of NaCl"? So I've made another lysis buffer with 200 mM NaCl. This was also with NiNTA purified. But the chromatogram is totally the same (attachment). So the NaCl is not the problem.
Could you maybe recommend where I could read about the choice of the lysis buffer?
Your buffer preferably should not have any EDTA or DTT if you are using Ni-NTA purification.
What type of cells do you have? What method are you using to disrupt them?
The problem may not be with the buffer or column. You have to be sure your protein of interest is in the lysate. I advise you run an SDS-PAGE of the lysate, and if possible a western blotting, to be sure your problem of interest is in the lysate before purification
thanks Victor Markus , I'm going to do SDS next Wednesday . Unfortunately it's really short project.
Adam B Shapiro , IPTG induced Rosetta/pLysS pET30a-His-RAD51.
1. 0,5 ml pellet + 5 ml lysis buffer
2. Sonication on ice (10 * 30s) pause 30 s.
That may be too much sonication, overheating the sample and cooking the proteins. With such a small volume of sample, you probably only need 3 cycles of sonication. Use a microscope to see if the cells have been mostly broken. Overheating can be reduced by putting the tube in a mixture of ice and water, rather than just ice, to improve heat transfer. Plastic tubes aren't very good for heat transfer. If you can find a stainless steel tube, that would be better.
Another thing to try is to take some of the cell suspension directly before sonication, add SDS-PAGE sample buffer, and run it on a gel to see if the protein is being expressed. Compare to an uninduced sample.
Finally, after sonication, centrifuge the sample at 5000 x g for 10 minutes. Check the supernatant and the pellet by SDS-PAGE to see if the protein is soluble (supernatant) or insoluble (pellet). A large, whitish pellet at this stage is a sign of the presence of inclusion bodies (insoluble protein).
EDTA will Strip your column of Ni2+, thus binding capacity is lost.
SDS page are in the attachments. We also made another experiment: SDS page for CVE (200mM NaCl lysisbuffer) with wash buffer without imidazole at all.
I don't see RAD 51 at SDS with high salt lysisbuffer ( it should be about 37 kDa).
I can see RAD 51 in the first fraction when we didn't use any imidazole for buffer A. This is probably because amino acids which naturally contain His on their surfaces are binding to the column.
Be careful when interpreting the presence of a band of the expected molecule weight on a gel, among many bands, as indicating the presence of the desired protein. It may be a different protein that happens to have the same molecular weight. You should confirm that it is the right protein by a Western blot, or by cutting it out of the gel and submitting it for mass spectrometry.
Adam B Shapiro Thank for your answers! It really helps to think in different direction about the problem. Unfortunately the project is finished, it was only for 3 weeks.
For information, when having any chelating (such as EDTA) or reducing agents (such as DTT) in your samples you should use a Ni-charged resin that keeps the Ni-ions attached to the resin also during these conditions. WorkBeads NiMAC resin and prepacked BabyBio NiMAC 1 ml and 5 ml prepacked columns with help you! I attached some information about these new tools that can be of help.
- Badges
- Science method