9 replies to this topic

[nirajm]

Dear All,
I am doing GST pull down assays. The GST fusion protein is not gets purified singly after incubation with the beads. And when I do the pull down using the in vitro synthesized protein, negative control (GST only) also shows bands. How can I stop the negative (GST only) from interacting with my protein. Any idea or protocol about running a preparatory gel is very welcome.
Hope to hear soon.
Thanks...

Edited by nirajm, 09 July 2009 - 12:49 PM.

[cellcounter]

nirajm, on Jul 9 2009, 12:30 PM, said:

Dear All,
I am doing GST pull down assays. The GST fusion protein is not gets purified singly after incubation with the beads. And when I do the pull down using the in vitro synthesized protein, negative control (GST only) also shows bands. How can I stop the negative (GST only) from interacting with my protein. Any idea or protocol about running a preparatory gel is very welcome.
Hope to hear soon.
Thanks...

You can make post pull down washing step more stringent. You can do this by using higher salt buffer (i.e. if you are using 100mM, do it with 300, 500mM etc), and if that does not work, by using higher conc of detergent (No detergent ->0.1% NP40 ->0.5% ->1%). For an IP using purified protein, generally higher stringency is not required, but that varies case by case.

[nirajm]

Hi Cellcounter,
Thanks a lot for quick response. I have used both the things. Prolong washes (five) with the high conc of salt. But I am unsuccessful. So, I think preparatory gel thing might work. The other thing is the very faint band in positive too. I mean I am not getting very think and fat band in positives. I ll appreciate if you have the  preparatory gel protocol.
Thanks again.

Edited by nirajm, 10 July 2009 - 08:37 AM.

[pras45]

Try preclearing the protein before moving on to real experiment, it could be that your protein is very sticky,, also as mentioned in the first reply ,stringent washing is also a good idea.

[nirajm]

pras45, on Jul 10 2009, 09:59 AM, said:

Try preclearing the protein before moving on to real experiment, it could be that your protein is very sticky,, also as mentioned in the first reply ,stringent washing is also a good idea.

U mean I should preclear the in vitro synthesized protein by immuno procipitation?
Thanks a lot.

[pras45]

nirajm, on Jul 10 2009, 10:21 AM, said:

pras45, on Jul 10 2009, 09:59 AM, said:

Try preclearing the protein before moving on to real experiment, it could be that your protein is very sticky,, also as mentioned in the first reply ,stringent washing is also a good idea.

U mean I should preclear the in vitro synthesized protein by immuno procipitation?
Thanks a lot.

ok, please be explicit on what you are trying to achieve here. Are you trying to purify your GST fusion protein using affinity choromatography (GST beads) or you are trying to do GST pull down assay, where you have a bait and a prey protein.

Your topic title and question is unrelatable. A little more info would be helpful.

[nirajm]

pras45, on Jul 10 2009, 09:31 AM, said:

nirajm, on Jul 10 2009, 10:21 AM, said:

pras45, on Jul 10 2009, 09:59 AM, said:

Try preclearing the protein before moving on to real experiment, it could be that your protein is very sticky,, also as mentioned in the first reply ,stringent washing is also a good idea.

U mean I should preclear the in vitro synthesized protein by immuno procipitation?
Thanks a lot.

ok, please be explicit on what you are trying to achieve here. Are you trying to purify your GST fusion protein using affinity choromatography (GST beads) or you are trying to do GST pull down assay, where you have a bait and a prey protein.

Your topic title and question is unrelatable. A little more info would be helpful.

Hi,
Sorry for the confusion. My main intention of getting purified GST-fusion is for use in GST-pull down.
Hope this ll make it more clear.

[pras45]

In addition to stringent washing, the precelaring step helps remove non-specific interaction to the GST beads. Incubate your prey protein with the GST beads, and take the sup and then use that for GST pull down assay. Make sure to run the precleared GST beads on gel too. Since you mentioned your prey protein is in-vitro syntesized, this means it is not pure and it could be in complex with some other protein that sticks to beads too.

[nirajm]

Thanks a lot Pras. I'll definately give it a shot. Thanks again for nice tip.
=
I have a one more question how do you grow and store the GST fusion protein ib bulk.
I mean do you freeze the sepharose bound GST fusion proteins at -80C till use? or after detaching them from Sepharose beads by reduced glutathione and freezing them?
Thanks a lot for your reply.

[OnceHere]

nirajm, on Aug 20 2009, 11:05 AM, said:

Thanks a lot Pras. I'll definately give it a shot. Thanks again for nice tip.
=
I have a one more question how do you grow and store the GST fusion protein ib bulk.
I mean do you freeze the sepharose bound GST fusion proteins at -80C till use? or after detaching them from Sepharose beads by reduced glutathione and freezing them?
Thanks a lot for your reply.

You can store the beads with 20~50% glycerol (+ your buffer) at -20C freezer, if you plan to use the GST fusion protein on beads for regular pull down assay.  Most companies sell these kinds of beads in glycerol.  But you do need to check the binding activity later on since some fusion protein might not be very stable in this way.