2 replies to this topic

[meri]

Hello everybody!
I have read a lot of topics about GST, Thrombin,...but I would like to be sure to do the experiment well, so I am here to ask you again some questions.

I am doing a GST-pulldown using GST-protX (26Da) and a mouse brain lysate, I want see 2 proteins that we will call A and B with molecular weight 55kDa and 24KDa, respectively. So, the problem is that I can't see the protein B because the MW is very similar with GST-protX.

I am thinking to use Thrombin to divide protX+bound proteins from the GST but...
1. Does Thrombin work well if there are beads in the solution?

2. I could eluate GSTprotX+bound proteins from the beads, cut with Thrombin and bind again GST with beads. Could be a good way to perform the experiment?

3. Do you know if I will see Thrombin in the gel after a ponceu staining? Because I don't know how many micrograms are one unit of Thrombin enzyme. I think I will use about 20units of Thrombin and I don't want to see a big band on the gel!!! I don't want to risk to cover the signal of my proteins A and B!!!

Thank you veeeery much for your help!

[meri]

hello again!
I wrote to GE healthcare to understand how many micrograms are 1 unit of Thrombin:

The specific activity is at least 7500 units/mg which means that 500 units corresponds to 66.7 microgram.

So If I will use 30units of Thrombin maybe I will load abot 4ug of enzyme and it will not give me problems!

[metionina]

I use 100 U of thrombin and it works well, with no problem for gel staining.
Your option 2 I think it's the better way to cut and then recover your protein.
There is also a thrombin (I forgot who sells it) that is bound to agarose beads: an easy way to  isolate the thrombin after digestion.