Hi,

One of the features of the GST tag is that it won't stick to the column if the protein is misfolded. Because of this, I would look at the induction conditions and other details in the protein expression process if the GST tag is not working for you. Specifically, I would try to slow down expression by lowering the incubation temperature after induction (I generally use 18 degrees), decreasing the amount of inducer, or inducing at a different cell density.

The salt is necessary for protein stability and should be left in your solutions. EDTA probably won't have a significant effect on binding to the column either.

If you saw the target protein in the wash fractions, but not in the elution, concentrating the elution fraction will most likely not be worth the time.

Good luck!

Thanks @Ryan Wheeler and appreciate your sgggestions. I agree with you and we always induce the LB after hitting 0.6-0.8 with IPTG and allow it to grow at 15C overnight. The issue I clearly noticed is that the supernatent flow through and the wash buffer is showing the GUS protein bands along with the other expressed proteins. My understanding is that the column is unable to hold the GST protein in the column and or unable to discriminate it from other untagged proteins. I also noticed that the bed volume of the resin is just 2 mL in a 10 mL column and the flow rate was relatively fast and certainly faster than 1ml/1 minute. I was increased the concentration of the reduced glutatione in the elution buffer from 10mM to 100 mM and absolutely saw no bands in the SDS PAGE, indicating that the GST bound protein is tethering to the columns well.

Either the target:

A. doesn't bind (as you see it in the flow-through); or

B. it binds and saturates the column, and then excess runs through (as you see it in the flow-through); AND then

C. it never comes off (as you don't see it in the eluent); or

D. the bound protein properly elutes but then something happens to it (as you don't see it in the eluent).

To check possibility C (see whether your protein sticks to the beads) you can boil a little resin in SDS-PAGE sample buffer and load the supernatant onto your gel. Check the resin after washing and after elution.

Now, let me go through your questions.

1. This is possible (my A, above), typically because of something weird in the sample or sample buffer. Double-check pH. Make sure there's no GSH in the sample (e.g., cell growth medium). No chaotrope or detergent.

2. That can't hurt. GSH is fairly cheap. Watch the pH. You can also include 0.05% Tween 20 to help break up non-specific stickiness (my B, above).

3. You can try that. It probably will reduce stringency of washing, which may give you more non-specific binding.

4. No. Don't leave out the EDTA. It won't inhibit elution, and i's important to inhibit proteolysis.

5. That shouldn't be necessary. Be sure to include protease inhibitors (in addition to EDTA) in the eluent (my D, above).