3 replies to this topic

[jeff kwak]

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

[mdfenko]

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

[chandra3316]

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

[doxorubicin]

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