are you sure your GSH working? what does that 10mM reduced Glu means, you should contain 10mM GSH in your PBS buffer.

Hi,

the reduced glutathione should be in Tris  buffer pH8 and prepared fresh. Check your pH of your elution buffer. 

Are you sure that 9000RPM is 500g in your centrifugation device? 9000RPM sounds aggressive for sepharose beads..

good luck

Hi Wang, I am sure about GST. Because I confirmed it by sequencing and found desired size band in SDS. I use 10mM reduced glutathione in elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0).

is there any chance you add too much elution buffer, so the protein was diluted too much to see in the SDS-PAGE gel, maybe you could try less volume elution or try Western blot with anti-GST antibody. good luck!

I would definitely check pH of elution buffer. It might happen that after adding elution buffer protein precipitates on beads. In lab we make larger amount of elution buffer adjust pH and store frozen. Mixing in 10 mM reduced glutathione in 50 mM Tris pH 8, might reduce pH down to 5-6.

Hi Julijus, I add reduced glutathione powder directly to 50 mM Tris-HCl pH 8 for making 10 mM reduced glutathione. Like 3 mg reduced glutathione powder in 1 ml 50 mM Tris-HCl pH 8 for making 10 mM elution buffer. All time prepare fresh buffer for experiment.

Yes, but did you check pH after the addition of glutathione?

Could it be that your protein is unfolded and binding to the beads aberrantly? Check the purification system with a positive control. 

Hi there;

2 possibilities : either your protein aggregates on the beads and therefore GSH is hopeless or the GSH binding site of your refolded protein is inaccessible to free GSH when interacting with GSH beads(then no competition and as a result no elution). Is there any proteolytic site present between GST and protein sequences? If yes have a go with on-column protease digest in order to free your protein. If it's not aggregated you should be able to recover it as a soluble object.

Hope it helps

Hi,

try with different concentrations of Glu. It may elute at one concentration.

@Reche, I will check by a positive control. @Liger, I have PreScission protease cleavage site between GST and protein sequence. I will also try it by column protease digestion.  

Hi Udaya, actually I Used GST fusion protein renaturation kit from cell biolabs (http://www.cellbiolabs.com/gst-inclusion-body-refolding). You can get the details procedure in kit manual. But I faced problem in elution of my protein. 

try to elute in 50mM Tris-cl (ph 9) along with up to 50mM reduced glutathione you may get elution

@shakhinur how did u eluted ur protein finally??  Iam facing same problem of elution

@Akanksha Pandey.  I met the same problem when I tried to elute GST-cleaved fusion protein, It seems my protein retained in the beads, nothing in the supernatant. I treated the pellet containing target protein with denaturation followed by renaturation, the protein was completely eluted. hopefully it will help you.   

Just came to this thread to check for reasons why my student couldn't elute his GST-tagged protein. Or Gertman above said 'check the pH of your elution buffer'. Did that. Was pH 3.3! My student had dissolved 50mM GSH in buffer pH8 and didn't re-adjust the pH.

Thanks!

If your protein is aggregating it might just not like pH 8. We've done GST-elutions at pH6 and it works just as good if it makes your protein happy!

Did you try to cleave the tag before going on the GSH beads? Than only GST binds to GSH beads. This is also one of purification possibilities.

Another point is to adjust the pH of your elution buffer. Use the pH your protein likes. Salt, glycerol, detergent can solubilise your protein if it precipitates on the beads.

Good luck

hi islam

the addition of reduced GSH to Tris-Hcl will lowered pH to acidic, so you should adjust pH 8 after adding GSH.

Use a high pH Tris-HCl buffer (8.0 - 9.0), supplemented with 20 - 50mM reduced glutathione. pH the solution again and readjust the pH to the required value and let the probe read the pH for 5 minutes from the time it gets stabilized. Additionally, you can supplement the GST-elution buffer with 100 - 150 mM NaCl and 1mM DTT if required.

@Tamer. You are right addition of reduced glutathione will dropped the pH to 3. You need to re-adjust the pH to 8 then add to the column to elute GST-fusion protein