I have been trying to elute protein of my interest tagged to GST using a protocol containing 10mM reduced glutathione buffered at pH 8.0. However, contrary to the proposed protocol for this elution to take place, I am having much less success as more than 95% of the fused protein is remaining bound to the beads (beads for binding to GST). I even tried to supplement the protocol by adding 2-mercaptoethanol so that glutathione would be kept at the reduced form. Still, the protein is not getting eluted. Can anyone please suggest the reason for this and any modifications that need to be done for proper elution from the beads?
You can try eluting with 20mM reduced Glutothione+5mM 2ME. If you still dont get enough, then you may try adding some triton X-100(0.2% may be) to distrpt hydrophobic interaction between proteins.
Apart from this, be sure you are checking the amount of protein which bound (add flowthough fraction on gel), eltution and bead/uneluted fraction on the gel to get an idea what is really happening.
Good luck.
I worked with elution/regeneration in SPR devices some time ago and by combing different chemical properties in one solution, we created some combinations that worked really fine. An acid supplemented with different kinds of ions was for example surprisingly good. All was published so read the story here
http://www.ncbi.nlm.nih.gov/pubmed/10405611
Hope that it brings some insiration. Good luck!
There are two possibilities either ur protein is aggregating on column or glutathione u r using is not good. Just try new batch of glutathione or try acid extraction method. I am concern about protein aggregation.
good luck
Edit: My memory (and mys student's) failed me - we also use 10 mM, I had a chance to check the protocol today.
(To elute GST fusion proteins, we use a lot more glutathione. I don't remember exactly, but it's between 100-300 mM.)
Amit's answer is most likely the case. You have protein aggregation or your glutathione is oxidized. Make fresh 10 mM glutathione buffer in 50 -100 mM Tris-HCl pH 8.0. You can add 100-500 MM KCL OR NaCl and 10% glycerol to help make the protein more soluble. You need to have at least 1 mM DTT or 2-ME. It is very important is to pH your glutathione buffer after you make it to pH 8.0. Do not make a stock solution of glutathione and store it for future use. You need to make this buffer fresh. If you use a ridiculous amount of glutathione of 100-300 mM you will need to have a much stronger buffer to maintain the pH. Good luck.
@ Michael-I have prepared everything fresh;had added the powder form of the reduced glutathione for making the elution buffer instead of adding from previously prepared stock; even tried to add salts as u mention but went only upto 150mM.I will try out with higher concentrations though.I agree with u that 100-300mM glutatione will be too high a concentration to maintained at pH-8.
Anyways,thanks all.
Hi Arnab, I had totally misremembered the concentration, we use 10 mM. And even with 10 mM, we readjust the pH after adding glutathione, as Michael said. Good luck!
I generally use 50mM. In some cases the elution peak will tilt a bit. This is generally solved by using 60mM.
Hi,
i have just eluted my protein from GST beads today
I have used 5mM GST but the pH of tris buffer was 9.
I think my elution has been around 70%
so, may be you can increase the pH to 9
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