I wish to measure ALP activity in cultured rBMSC. I found few methods to obtain cell lysate using RIPA.
I) scrape out the cells in PBS, pellet the cells and treat in RIPA
II) Trypsinise the cells, pellet and treat in RIPA
III) Directly add RIPA onto the attached cells
Which would be the better option? I would also wish to know if there is any other lysis buffer works better in terms of ALP assay.
Hi Sreekanth. Cell lysates are horrible to work with. What I did when I was doing western blots was to treat a plate with ~3 ml of RIPA buffer and use a syringe with a small bore needle to suck up and dispel the contents about 10 times.
This sheared the DNA and made it possible to pipette accurate amounts without the snot-like consistency of raw cell extracts.
Hi Sreekanth. I don't know if you have to use RIPA buffer for ALP assay. We use 1x Triton and freeze- thaw the samples thrice to extract cellular content. It worked well for ALP and DNA assays. If the cells secrete a dense matrix, more rigorous pipett ing and sonication should do the job of getting the cellular contents in solution.
Best,
Aja
Hi Aja, Thank you for your suggestion. Do you pellet the cells and then add buffer
?
Hi Sreekanth,
We just add the Triton buffer to the cells as such, followed by the freeze thaw cycles.
I would look into the data sheet of your assay kit though, just to make sure that your kit is compatible with the buffer system.
We use a kit from Bio-Rad.
Good luck with your assay!
Best,
Aja
Easiest way will be to use 1% Triton X100 in PBS for 30 min in RT
Dr. Bhowmick, Dr.Aja
Do you use Protease Inhibitor Cocktail or PMSF in the buffer?
Hi Sreekanth, we usually use M-PER (mammalian protein extraction reagent) and works very well. This buffer completely lyses cells in few seconds (directly on attached cells) and prevents protein denaturation, hence retaining protein activity. In addition, is compatible with several protein quantification methods such as BCA and Bradford.
Hi Sreekanth,
ALP assay is pretty simple as far as my experience has been. We don't use Protease inhibitors. I would however make sure I adhere to the assay kit's protocol. Our assay was based on a substrate digestion rate by the enzyme, and did not require anything other than a surfactant based cell extraction.
Good luck with your experiment!
Best,
Aja
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