cytochrome c western help - (Jun/02/2008 )

Hi, i've been trying to detect cytochrome c in my whole cell lysate (HeLa) via western and i've got these 50-60kDa bands instead of the expected 12-15kDa.

Is this normal? I'm running a 12% gel. after transfer i dont see any low kDa bands at all. I'm using the invitrogen tanks, transfering at 30V for 90 min. Is it possible the the cyt c has moved pass the pvdf into the filter paper?





Thanks,

cheers.

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It's not probable cit-c are passing through PVDF membrane. About band on 50 kDa... are you boiling your sample before loading in gel? Is it a denaturing gel with SDS?

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Are your bands of 50-60 kb strong on your membrane?

I am working on HeLa and Cytochrome C as well. haven't done it yet, but soon will do.

Invitrogen tanks of semi-dry are you using? I use Biorad for 15 V and 60 mA for 1 hr.

Do you see 12-15 kb blue bands on your SDS-PAGE gel? if you see those bands then probably Cyt C must be there, but need to repeat with a concentrated protein to get them on western too.

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Dude I just saw something and wanted to let you know, my friend uses 1:150 for Cyt C antibody........he says for Cyt C he needs to use higher amount of antibody.

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How much protein are you adding to your gels? I've been having trouble recently detecting the lower kDa bands too. I've now check and realise that to get good detection you need to be adding at least 30-40ug protein per sample or you just won't see them. (We use primary at 1:1000 still and incubate at 4 degrees overnight!)

B

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Hi all, thanks for the input!

I've been trying again and again and i still don't see the band 12kDa band~
And then the 50kDa bands disappeared too!

I'm running denaturing SDS-PAGE, boiled the sample before loading. And because i'm doing some protease shearing assays (using proteinase k and triton-x to solubilize the membrane), i don't really know how much is the final protein concentration. But my starting material is 50 to 100 ug.

I'm doing wet transfer. Tried transfering between 1hr to 1.5hr at 30V. Still no progress and it's been 3 months!

I'm also looking at other mitochondrial protein controls, such as Hsp70 and MFN2 and they all worked brilliantly.

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use less protein, use 20kg

if you load too much you'll get smears and your proteins can not run through the gel. I added 30ug, 68ug and 320 ug last week to check the intensity of my bands but I could only see visible bands with 30ug.....over 30 ug you'll end up with smear.

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