western blotting of DNA-binding protein of mammalian cell

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drelamuruganvet
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western blotting of DNA-binding protein of mammalian cell

Post by drelamuruganvet » Mon Jan 19, 2015 9:39 am

i am trying to immunoblot one of the DNA-binding protein....
first i have cultured the cells in 25 cm2 flask and collected the cell lysate using RIPA lysis (1 ml) buffer...kept in ice for 5 min, followed by passing the lysate through 24 G needle about 5 times...then centrifuged at 12000 g for 15 min..collected the supernatant and pellet..
from the supernatant 40 ul of sample taken and 2X sample buffer added; to the pellet 50ul of 2X sample buffer added, boiled for 5 min in 95C and SDS-PAGE...
duing SDS-PAGE in not getting a banding pattern instead i got full smear of gel....simultaneously i have added other sample they ran properly but not this cell lysate...

what could be problem..how to get a clear SDS-PAGE and western blotting....

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