please help with my western blots! - (Mar/01/2013 )

hi,

i am running western blots on tissue samples, specifically pancreatic tissue samples. I homogenized my samples in 1 mM EDTA, 0.5% TritonX in PBS (Ph 7.4) with protease inhibitor. After that i spun down the samples at 1000G for 3 mins and found the concentration of the supernatant with BCA. The problem occured after reducing the protein samples. btw i am using invitrogen's Bolt apparatus and their supplies to reduce the samples. probably just their own version of laemmli buffer. anyway, the reduced samples are very jelly. i could not load them in the wells because they kept cling onto the tips. and those samples that i was able to load came out jagged. the blot ran straight, but instead of nice flat lines, i see jagged lines; almost in zig-zag like!

I am thinking that my samples are dirty. I only spun at 1000G for 3 mins which makes me believe that there are a lot of junk in my trunk. do you guys think that spinning it down at 14000G for 15 mins would help?

I am thinking about using DNase and RNase. when i isolate RNA, things get very sticky. mayhaps it's RNA/DNA jumk in my trunk? what do you guys think? oh yea, and if you think this is the problem, how much DNase/RNase should i use?

I dont believe in god, but if you are up there, please help me superman! - homer simpson

thank you.

my best,

jeff kwak

it's probably genomic dan. you may be able to loosen it up by vortexing vigorously or sonicating or treating with dnase.

Lyse the cells in the liquid nitrogen.. that will avoid jelly....:)

mdfenko is right....it's definitely DNA. Find a probe sonicator and shread that DNA! Wear ear protection and close the door!