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protein extraction from pirmary keratinocytes

extraction keratinocytes western blot

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#1 EvilTwin



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Posted 03 January 2012 - 03:24 AM

I've been trying to isolate proteins from human primary keratinocytes using RIPA buffer, but every time i get very low concentrations (ranging from 0,8 ug/ul to 1,5 ug/ul). I do it by trypsinising the cells, harvesting them to an eppendorf tube, centrifuging and suspending the pellet in RIPA.

I have never encountered this problem before with other cell lines.

I've modified the the protocol I had been using (changed the centrifugation speed from 1.200 rpm to 2.000 rpm, use different eppendorf tubes - the result improved a bit, but is still insufficient). The low concentration results in huge solution volume I need to apply on electrophoresis gel and I think that it causes problems with protein detection (with HRP-conjugated antibody).

Do you think that this low concentration may be really the cause of detection problems (uneven, dot-shaped or beads-shaped bands)? I use high antibody concentrations and long exposures to detect my protein of interest, and when it shows, the blot looks quite awful and is not suitable for any interpretation.

Do you have any ideas how I can improve the protein concentration?

#2 EvilTwin



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Posted 13 January 2012 - 03:37 AM

Can noone help me?

#3 mdfenko


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Posted 13 January 2012 - 06:31 AM

it's the high volume of salty buffer rather than the low concentration that is causing the problems with the gel and blot.

you can either reduce the volume in which you prepare the proteins or dialyze the sample to remove the salts or both.
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