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please help with my western blots!

western blots tissue lysate

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3 replies to this topic

#1 jeff kwak

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Posted 01 March 2013 - 03:12 PM

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

#2 mdfenko

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Posted 01 March 2013 - 03:35 PM

it's probably genomic dan. you may be able to loosen it up by vortexing vigorously or sonicating or treating with dnase.
talent does what it can
genius does what it must
i do what i get paid to do

#3 chandra3316

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Posted 01 March 2013 - 09:46 PM

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

#4 doxorubicin

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Posted 02 March 2013 - 03:12 AM

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





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