hi I am having a frustrating time harvesting human articular chondrocytes to run on a Western. I am harvesting 2 x 6 well plates by gentle scraping in PBS, spinning down then resuspending the whole lot in 100 uL RIPA or Tris/NP-40 buffer. the lysates are then frozen and then next day thawed on ice, spun 12k 10 min (4 degrees) and the supernatant kept - but even at this point there is very little pellet of insoluble protein. From two wells I am getting very little protein (
The volume of RIPA is too low, makes the buffer unable to lyse and soluble all the cells. Try using protease inhibitor cocktail and increase your incubation at 4C (at least 30 min by shaking).
Directly scrap and treat cells with SDS PAGE sample buffer that contains SDS. Centrifuge cell debris and load supernant after heat denaturation on gel for electrophorisis. This shall work.
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