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gelatin staining with coomassie: how do I make it work?


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#1 mascat

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Posted 11 April 2011 - 02:46 AM

Hi everybody,
I have been trying to set up gelatin zymography in my lab and ran into a strange problem. When I previously stained gels with a really old and dark Commassie solution somebody once gave me, the gelatin inthe SDS polyacrylamide gel stained nicely, giving a dark blue background over which I could see MMP digestion bands. When I ran out of this solution, I bought Bio-Rad Biosafe Coomassie, just to find out that no matter how long I incubate the gel with it, it will stain the protein bands but it never stains the background, making it impossible to discern any digestion bands...
Any suggestion as to why this happens? Why would gelatin not be stained by this type of Coomassie?! (I use 300 bloom gelatin at a final concentration of 1 mg/ml)
I am willing to buy a different Coomassie, but which one would be best to overcome this problem?
Thanks!

#2 protolder

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Posted 11 April 2011 - 09:58 PM

Hola,the difference is that the lended solution is made with c.blue acetic acid and methanol, it stains blue all the gel and after you need wash with 10% acetic acid to clear a bit the blue background. The commercial biosafe c.blue has phosphoric acid and itĘs designed to stain protein bands withouth stain the bacground. So prepare the conventional staining solution with c.blue acetic acid and methanol, wich you can reuse, and prepare acetic acid 10%wich stain of blue as the gel clears and you can decolorate and reuse with active charcoal or some pieces of foam rubber. The stain recipes are in the net,probably in any of the protocol on line of this page or in any lab manual as Maniatis. Buena suerte

#3 mascat

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Posted 12 April 2011 - 01:07 AM

That makes a lot of sense! So I need to buy the powder and make the solution that way ... Thanks a lot!!

#4 mdfenko

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Posted 12 April 2011 - 08:32 AM

the commercial solution may have been made with coomassie brilliant blue g-250 and your friend's may have been made with r-250. g-250 is more specific than r-250 so the r-250 will stain more.
talent does what it can
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