RIPA Buffer and DNA - (Mar/06/2008 )
As I'm lysing endothelial cells with RIPA buffer containing 1% NP40, 0,25% Deoxycholate and 0,1% SDS, I get very visquous lysates but only sometimes and I do not get why this happens so randomly!! Someone has a clue??
I know that RIPA buffer disprut nuclear membrane and release nuclear proteins but viscosity is due to the DNA; Why dos this viscosity appears only sometimes?
Thanks for your help
Can you correlate the visquosity with a large amount of cells vs a small amount of RIPA?
I always lyse my cells in a RIPA-like buffer, and since I always have the same amount of cells and use a constant volume of RIPA, I never experienced visquous solutions.
Hope this helps!
I always lyse my cells in a RIPA-like buffer, and since I always have the same amount of cells and use a constant volume of RIPA, I never experienced visquous solutions.
Hope this helps!
Hey! I am having trouble as well with viscosity after lysing. This is my RIPA protocol:
1L= 8.76g NaCL, 10ml 1M Tris (ph 7.2), 10ml 10% SDS, 1ml Triton-x 100, 10g Deoxycholate, 10ml .5M EDTA, bring volume to 1ml
I thinkm this may be too strong and I am getting DNA. How can I dilute this?
As Madrius said, the viscocity probably correlated with your cell number, i.e. amount of DNA.
Scrape your cells in RIPA buffer, incubate for say 10 min on ice, then spin 5 min at around 12 000 g (top speed of bench top centrifuge) at 4C. This will compact the DNA etc, so you can collect the lysate. If the pellet is very big or still very fluffy (rather than pellet) then you need to increase lysis volume.
IQ, the usual detergent conc is 0.5-1% NP40 or Tx-100, 0.5% DOC, 0.1% SDS.