My aim is to phosphorylate the proteins (produced by rabbit reticulocyte lysate) via a specific kinase and then remove the phosphorylation via lambda phosphatase (LP). The problem is before LP treatment, proteins of interest and kinases are all dissolved in the buffer. I am afraid the remaining kinases will affect the reactions later. So I want to remove the kinase, and of course not by centrifudge. Then I boiled the samples after kinases treatment on 65°C for 20min. After boiling, there are a lot of red pallets deposited in the bottom of the tubes. Is anyone know what are these pallets?
In my in vitro phosphorylation assays, there are ST buffer, ATP and protein lysates in the tubes.
Dear Yuling, heating/boiling will denature most proteins in your assay, hence the precipitate.
Ideally, you will be able to immobilize your kinase onto beads (magnetic, sepharose, etc.) so you can easily remove it after the assay. If you have the pure kinase available, it might be easily done with carboxy activated beads (NHS/EDC chemistry), otherwise you could immobilize antibodies against the kinase in that way and use the beads to fish for your kinase.
If you are very lucky, poisoning the kinase with vanadate, arsenate or fluoride might work, too, but there is quite a risk that this will also inhibit the phosphatase later.
Edit: But you could try to remove these inhibitors by gel filtration/desalting (e.g. PD10/Sephadex G-25)
Dear Yuling,
I agree with Wolfgang.
To avoid the remaining kinase action later did you think of adding a kinase specific inhibitor if known? Of course you need to set up a reaction kinetic, check the working concentration, etc.
Good luck,
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