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SDS-PAGE Cell lysis problems


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#1 andyginna

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Posted 23 June 2009 - 08:11 AM

Hi!

I am trying to resolve lysed mesenchymal stem cells extracted from bone marrow cultures on SDS-PAGE gels (Res=15%; Stack=5%). I have used these gels for serum before successfully, but am unable to resolve any bands from cells after staining with Coomassie Blue. I am reducing lysates in sample buffer with 5% 2-mercaptoethanol and denauturing at 100C for 5 minutes prior to cooling samples on wet ice. The loading is ok (7ul), and the tracking dye migrates uniformly during electrophoresis via Laemmlie.

I am just wondering why I am not getting any bands other than the protein marker standards. Do I need to use more cells? I used about 5000 (five thousand) per well.

Any advice would be greatly appreciated!

Thank you

Andy :)

#2 mdfenko

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Posted 23 June 2009 - 12:20 PM

you may want to try a more sensitive stain, like colloidal coomassie or silver stain.

since you are using a whole cell extract, are you looking for any higher mw proteins?

a 15% gel will only allow smaller proteins to resolve well.

on the off chance that you are causing your proteins to aggregate by overheating, you can try heating at 65C for 10-20 minutes.
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#3 andyginna

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Posted 23 June 2009 - 01:11 PM

you may want to try a more sensitive stain, like colloidal coomassie or silver stain.

since you are using a whole cell extract, are you looking for any higher mw proteins?

a 15% gel will only allow smaller proteins to resolve well.

on the off chance that you are causing your proteins to aggregate by overheating, you can try heating at 65C for 10-20 minutes.



Thanks mdfenko, I'll give it a try. Andy

#4 Aris

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Posted 24 June 2009 - 05:16 PM

What you mean 5000 per well??? Cells??? What amount are you loading? The Coomesie stain takes about 30 min to stain and 30 to destain and it is a mess. Ponceau S is better and more sensitive.
You talk a lot about your running settings. What about transfer? What transfer are you doing? How does you gell look like after the transfer? Have you stained it?
These are all very crucial information, please try to be more specific




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