I would like to measure the activity of PP2A in neuroblastoma. So far I have tried many experiments and I am not able to get a very good activity. I am using the PP2A immunoprecipitation phosphatase assay kit from millipore. I have treated my cells with increasing concentrations of a PP2A activator for 24h. I extracted protein according to the kit protocol and used supernatants for enzyme assay. I get a slight increased activity with increasing concentrations and I am not satisfied because in the literature, they show better activity than mine. So does anyone has suggestion how to improve it?
1- How to improve the lysate preparation? maybe something during protein extraction already affects the enzyme activity. I handle my samples on ice.
2- in the untreated control, I have already a high level of free phosphate (400-600 pmoles of phosphate) and the gain of activiy in the treated samples is around 200-300 pmoles maximum, compared to the literature where they get at least 2-fold increase in activity compared to control.
3- is it always necessary to normalize the enzymatic assay with the IP?and if yes , how should I improve and make this IP? meaning how much should be loaded (same volume use for the enzymatic assay, knowing that if I use the supernatant for IP after elution, it is difficult to know exactly how much you have loaded?)
before the measurement of activity, the protein mixed with buffer, beads, enzyme are incubated at 4°C for 2h in the constant rotating motion instead of rocking motion as recommended by the kit instructions. Do you think it is enough time? also is 24h treatment to much for the treatment or to less? knowing that highest concentrations of the PP2A activator kill the cells within 24h? but still at lower range of dosage, the cells look OK
4-the protocole provided by milliopore does not describe any elution step, so I don´t measure the free phosphate in the supernatant rather I measure it in the bead fraction
5-sample storage: sually, I always perform the assay directly after protein isolation and calculation of concentrations, and I store thereafter the remaining samples at -80 °C. Is that OK?
Thank you all...
Hi, do you use phosphate buffers in any of your steps? Phosphates can inhibit phosphatases.
Hi Steingrimur Stefansson
In the kit there is actually a pNPP Ser/Thr Assay buffer. So it is then possible that I replace this buffer with Only TBS? the pNPP is suppose to enhance the phosphatase activity, so in that case what reagent should I use as an enhancer?
Thank you
Hi Célimène Galiger ,
Did you find any clue to resolve this problem or you stop using this kit?
Please let me know. In some experiments, I also have the same experience as you regarding this kit.
Has anybody had any luck with this kit? There are a couple of publications indicating this kit suggesting issues with this kit. I run some key control experiments and it certainly does not work with me I raised my own issues with Millipore but they refused to refund me or even taking the kit down. Please get in touch!
Hi Mary, do you know if the activator is reversible or irreversible binds to PP2A? if it is reversible, likely the activator would be diluted and removed during IP/ wash step... It is common for most kinase/ phosphatase assay. That's why when you finished all IP/ wash step and do the readout, the result is not as expected. If you remember, just like ATRA binding to PML-RARA, it is reversible and to maintain the PHF8 binding to ATRA-bounded PML-RARA, Mari-Frances needed to add ATRA to the cell lysate when performing the IP. So you may need to do something similar.
If you have known PP2A target of your cells, may be better do the WB, it is even more physiological.