hello to everyone, I would like to let you know that I have solved the problem, by incubating the resin with PBS containing 10mM DTT (final concentration) for 30min at RT in a rotating plattform (so the resin is kept in suspension) and after that my protein did elute, while the GST moiety remained bound to the resin. Thanks to everyone for your help!

As per my opinion you ma try to increase(/decrease) the salt conc. of elutant or change the pH. Even if danaturants are not working then I am completely agreed with Krzysztof Wicher that protein might have high affiny towards resin or GST, which may be test by incubating resin with crude glutathione agarosa and probably doing western.

This is a common problem. You can investigate protein solubilising agents in the literature. However, in my experience, the problem is most usually associated with instability of your protein once the GST tag is cleaved. Have you tried eluting without cleavage - that would test affinity of your protein for the beads. I now tend to think that the GST tag can sort of artificially stabilise or solubilise a protein that will simply crash out of solution once you remove the tag - however, you might be lucky and find conditions that give stable cleaved protein.

Hi...just try to collect the flow-through after thrombin digestion (the buffer+ thrombin that u use for in-column digestion)...it must have your protein...it will be diluted, so u have to concentrate by amicon filters. You will not get anything from the column (the way u are trying to do now) after u remove the thrombin buffer mixture, except from GST itself.

Are you collecting the flow through after thrombin digestion or are you adding elution buffer afterwards? Your protein should be in the flow through. If you are indeed collecting the flowthrough but your protein is still not there, it means it is binding to the resin or to GST. In that case, try increasing the salt concentration to 500mM or try adding detergent to your buffer (should eliminate any unspecific interactions).

hi Lucia,

You can try everything that has been written by other colleagues but if even though you don't get what you want that is because the site is not exposed. Which site? I'm talking about the enzyme thrombin. Thrombin must to recognize a site and then do the cleavage. If the site is kinda hidden the digestion is not going to happen. A possibility to solve that is: If you have cloned GST-ProteinX... why don't you try ProteinX-GST. Maybe this will solve this issue. Hopefully.

Best wishes,

@lex

Here’s a chart of GST elution problems you might consider; elution buffer pH and flow rate may need adjustment. They say you may need to use up to 50 mM DTT. http://www.abtbeads.com/literature/purification/gst/glutathione_beads_troubleshooting_guide.pdf

Johanna and others warn that under proteolytic conditions (temp., etc.) you could be breaking up your molecule; test that what is sticking is really the whole protein. And do you centrifuge debris and only use the supernatant? Don’t sludge up the column…

For protein-specific problems, first test the GTS column with a protein you know will elute and check that your system is working—perhaps the reducing elution buffer was off, or a bad column batch. If successful, then try conditioning the column with serum (which has a variety of proteins that are very hard to elute) then thoroughly elute and wash again—test staining some of the packing with Coomassie would show you residual protein that didn’t come off. Then I’d try running the test lysate on the conditioned column—maybe the conditioning will keep that affinity from being so strong. If conditioning is working, then switch the conditioning agent to a pure protein such as albumin (or whatever works) to keep the system quantifiable.

You could also use the column packing as a getter and dissolve with agarase (DNA fragments can be purified from agarose gels this way), but let’s hope the other steps (like stronger DTT elution) worked before resorting to that.

May be your protein becomes insoluble and precipitates, remaining trapped in the column. You should know the characteristics of you protein

Yes - my favourite suspicion would be protein instability - it is precipitating once the tag is cleaved. That is what I was getting at in my previous comment. However, it is early days of testing for you - so you might not have this unfortunate problem!

try to use 8M urea

Did you check the flow through? Proteins do usually run away from the column when they are not happy in solution.

I would add:

a) Thrombin site not exposed

b) Thrombin activity is poor under your conditions (watch out for DTT, BME. Obviously PMSF and other serine prot. inhibitors)

c) If your cleaved protein is enjoying some hydrophobic interaction with the resin, try adding ~ 10-20% ethylene glycol to your buffer and check the flow through.

HTH

Many thanks to everyone for your comments.

Some replies to your questions:

- yes, I collected the flowthrough after thrombin digestion, and then I did several washes with PBS, but in these fractions I recovered just little amount of my protein (size corresponding to the cleaved protein). Most of it remained in the column (I checked this by SDS-PAGE of an alicuot of the resin, and I see there two proteins: one of the size corresponding to GST (aprox 27kDa), and the other with my protein size (51kDa). So it has been cleaved from the GST).

- then I tried washes of the resin with PBS containing 0,5% Tx-100, and then with PBS containing 10mM DTT, but after all of this, very little of my protein eluted, and instead most of it still remained in the column

- I also tried to elute the protein from the column with a buffer containing excess of free glutathione and indeed it eluted (with the GST as well). So one possibility is that it remained bound to GST even after thrombin cleavage

I will try to increase salt concentration, or detergent concentration, or change pH. 10mM DTT is the maximum concentration that does not affect the binding of GST to glutathione, according to the Glutathione agarose brochure

More suggestions?

Thanks again!

Use reduced glutathione to elute the protein and then perform the digestion and then use batch (not column) removal of GSTs (that are cleaved off from your protein target). Otherwise, the trypsin you used might be dead, so that the protein is not eluted.

What appears from the above discussion, to me, is that the cleaved protein is getting bound to either the column or the GST moiety. In this case only increasing the detergents' concentration or ionic strength of the buffer can help you. If it is still not getting eluted you can elute both GST and your protein of interest via reduced-glutathione and then carry out a second step purification e.g. Gel filtration or I-E chromatography. According to me, GFC will be better if there is huge difference in the size of the native form of the two proteins. (I am not aware of the probable oligomeric state of your protein and GST is known to form dimers!). I hope this helps.

Good Luck!

I am also agree with Johana and tripathi comments. First protein elute without thrombin digestion with changing pH, and salt or buffer molarity to make sure that your protein could not bind to the GST agarose. It is likely possible to increase concentration of your protein in the column after washing it trend to partially misfolded or aggregate in the column.

good luck

hello to everyone, I would like to let you know that I have solved the problem, by incubating the resin with PBS containing 10mM DTT (final concentration) for 30min at RT in a rotating plattform (so the resin is kept in suspension) and after that my protein did elute, while the GST moiety remained bound to the resin. Thanks to everyone for your help!

Good you solved the problem! For the future: if a protein is insoluble without the tag, an addition of glycerol or sucrose to the buffer might help stabilise it, as well as high salt concentration. Anything you add must suit the later use of the protein, though.

Hi Lucia,

I met exactly  the same problem with my protein. So I am wondering what protein you were working on so that I could make a good comparison with my condition. I know this is an old story, but I am really appreciated if you could recall some details of your method, like the pH of the PBS. Thanks.