GST pull-down:Is there any way to reduce nonspecific binding of protein to gluta - (Oct/03/2007 )

Hi all,

I have a trouble to get specific interaction of two proteins.

I did GST pull-down assay to see if my GST-fused protein really binds to another protein in the cell lysate. After GST pull-down, I eluted protein complexes by boiling

with laemmli buffer, not by adding reduced glutathione. Then did SDS-PAGE and immunolotted.

The problem is that there is also a band of equal intensity in GST control sample. What can you recommend to reduce this nonspecific band?

Is competitive elution using reduced glutathione better than boling elution to reduce nonspecific band?

I heard that BSA blocks nonspecific binding of protein to beads. Is it true? Then, can anyone tell me how I can use BSA for GST pull-down experiment? What step do I

have to add BSA?

FYI, I used precleared the cell lysate with GST+glutathione agarose beads.

Great thanks to you in advance!!

I do lots and lots of GST pulldowns. First, I bind the GST-protein or GST to the glutathione beads, wash profusely and then block overnight in 5% BSA. You need to make BSA fresh and I filter mine to get rid of any precipitants which can destroy your results. Wash the beads twice in buffer to clear out unbound BSA and then incubate in cell lysate. I never precleared but it wasn't neccessary for me. If the band in the control is your protein of interest, preclearing will just remove the protein from the lysate and there is nothing left to test the interaction with. What buffer do you use? I use TIF (150mM NaCl, 20mM Tris (pH8.0), 1mM MgCl2, 0.1% NP40, 10% glycerol). How long are you incubating the beads/lysate? Try shorter times. I've gone as short as two hours. Finally, the last washes. You can increase the detergent and/or salt to disrupt nonspecific binding. This should help clean up the GST control. I would recommend a large scale pulldown which you can split up in the end and try multiple washing conditions. Also, try more washes with each wash rotating at 4degree for 10-15 mins each. Last but not least, I use an insulin needle to aspirate the wash buffer. This way I can remove all the buffer but not worry about loosing the beads. This makes washes much more efficient.

Competitive elution will help only if the protein is non-specifically binding the bead and not GST. Try incubating just beads with the lysate. If you see the protein with just the beads, competitive elution may help. Otherwise, I just boil the way you do. Good luck and I hope this helps.

Hi,

I have the same problems with my GST Pull-down assay. I get the bands as big as my protein in negative control that is beads bound GST. I was wondering is there any human or viral, or even bacterial protein except the GST substrate, which binds to GST?

Many thanks
P.S. did you solve your problem?

One thing we always did was wash the initial GST fusion prep with buffer containing 10% glycerol, which stopped most of the non-specific binding.
the comments about fresh BSA seem good for the next stage.