Dear All,
I need a protocol for estimation of protein from cell cultured in a 96 well plate. I want to lyse the cells in the same well and then do a protein estimation (preferably in the same well). Please tell me what volumes of lysis buffer should be good enough for ~40,000 cells in a 96 well plate.
I use Pierce™ BCA Protein Assay Kit for test tube estimation of proteins. If I want to use this same method, what ratio of reagent to lysis buffer should i choose? I am limited by the size of the well !
Thanks !
Parijat
A very effective and simple quantitative method is the Bradford Assay.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf
Good luck.
Parijat, in order to achieve a complete lysis (i.e. to reach all cells in the well), you might need to add 50..100ul of lysis buffer. And you'll probably have to mix (e.g. by pipetting) that all cells are lysed, but I'd try to avoid it as it will introduce significant errros when you analyze the material in the original plate due to uncontrollable losses in the volume i.e. the samples.
I'd try to set up a standard curve by providing the same volume of protein standards in lysis buffer and then add a practical volume (100..200ul) of BCA reagent and check if the protein amount is measurable. The standard curve doesn't need to be linear, as long as you can do a (not too weird) fit, like 2nd order polynomial or 4 parameter logistic (use e.g. Sigmaplot, Sigmastat or QtiPlot if your spreadsheet doesn't provide it). If the protein should be too much, just use 10ul or so in a separate plate.
BTW, thermocyclers and PCR plates are perfect for this kind of assay, so I'd actually opt for the 10ul approach.
Hi, I would go with a combination of both, lysis buffer and the Bradford assay (as already mentioned by Mr. Schechinger and Mr. Salih). First, chekc that the lysis is complete and if not, adjust the lysis buffer concentration (check with OD and/or microscopy). Then simply add the appropriate amount of Bradford reagent and give it a try. Should this work, perform a standard calibration with a specific amount of e.g. BSA to account for any side effects. I assume you use a plate reader? If your reader can shake and incubate, use these features for lysis and mixing, if not, use a standard shaker. All the best
Dear all,
Thank you for all suggestions, it was really helpful.
I lysed the cells in 96 well plate and then transferred the contents into a 24 well plate for protein estimation.
Parijat
I also want to measure protein levels of cells attached to a 96 well plate. If the lysis buffer is added to the plate itself, is it alright if the cell debris remain in the plate (if any remain after lysis) and bradford reagent is directly added into the wells? Or is it necessary to centrifuge out the cells after addition of lysis buffer to the wells?
Hi Parijat,
What volume of lysis buffer did you add per well of a 96-well plate?
Have you tried to directly perform the BCA assay in the original experimental 96-well plate, instead of transferring to a 24-well plate?
cheers.
But then are you guys just estimating the protein or using that for further western blotting..?? and also how much was the concentration you were able to get of the protein??
- Badges
- Science topic