HI all, Has anyone idea for protease inhibitor for Raw cells?
I am making cytoplamsic and nuclear fraction using PIERCE Niper cytoplamsic and nuclear kit. I am making fractions at four different time intervals(6,12,18,24) of raw 264.7 infected with H.pylori 26695 and I use to check expression of p50. I used to add 1x of 100 x sigma protease inhibotor but one of its constituent carried p40 inhibitor itself, so I am getting mutiple bands.
I don't understand it is cell line specific or of my desired protein?
It will be very helpful if someone can recommend me concentration too?
THANKS
Hi'
I think the best inhibitor of NF-kB is the Curcumin. Curcumin is very safe and easy to use.
Hi!
I normally use Pierce protease inhibitor (100x) supplied by Thermo. There are many reasons that could explain those multiple bands. Try adding a reducing agent (such as beta-mercaptoethanol) to your sample loading buffer. Have you checked if your primary antibody could be detecting several isoforms of p50?
Best wishes,
I have tested several cocktails and even develop my own voodoo protease inhibitor until I found the Pierce protease inhibitor (100x) supplied by Thermo advised by Teresa. I have with this PIERCE cocktail extracted protein from different cell lines, tissue and tracked protein phosphorilation in different cell fractions. NF-kB is tricky to tackle because of its different sub-units p50 and p65. In a denaturating gel normalize protein concentration in Lameli reducing agent with beta-mercaptoethanol (as Teresa says) and check on the same membrane p50, p65 and p105. It is always a beautiful work to play with NF-kB.
I hope this helps and happy new year.
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