I have seeded keratinocytes in 6 well plate. 120 ul of RIPA buffer(with Pro. K) was used to break cells on ice. However, Bradford reagent (Biorad, diluted with water , 1:4) did not develop any blue color upon addition (that is usual case when protein is present). cells were 100% confluent at time of lysis.
What could be possible reason of RIPA buffer failure. thanks
Hi Khuram,
Could you explain your process a bit more in detail? And what you are using for your RIPA buffer? Depending on cell type, addition of RIPA won't be enough and agitation/scraping of the cellular material off the plate may be necessary (with sticky cells). As well, highly confluent cells do not lyse with RIPA as well as just sub-confluent cells (95%).
Considering above, the detection limit of the Bradford assay in conjunction with your lysis condition might not have been ideal.
Try following the lysis protocol below (see BioID methods section) and seeing if there is a difference in what you're doing and here. For on plate lysis, add the (mod)RIPA directly to the wells, agitate, wait 5 mins, and "top-up" the SDS concentration to 0.4% - 1% (to whatever SDS conc. the Bradford Assay kit you are using will tolerate). This should be more than sufficient (with pipetting off the lysate thoroughly from the wells) to lyse your cells properly.
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