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protein extraction from human tissue (very small) to do ELISA


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5 replies to this topic

#1 Surya r

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Posted 07 September 2012 - 02:46 AM

Could anyone please provide the protocol to extract protein from the tissue that can be quantiifed by ELISA. Please also add the suitable tissue collection method (except snap freeze).

Many thanks

Surya

#2 Curtis

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Posted 13 September 2012 - 10:56 PM

These methods have been posted here before many times.. Just use the search button on the top right corner.

#3 Surya r

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Posted 20 September 2012 - 01:14 AM

Thanks Curtis. I searched but couldn't find any method that can be used to do ELISA. Could you please help me

#4 bob1

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Posted 20 September 2012 - 01:21 AM

Any of the protein solubilization protocols for western blot from tissue should work - ELISA is essentially the same process, you'll just have to scale down the reagents to the amount of tissue you have.

#5 Surya r

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Posted 20 September 2012 - 03:53 AM

Thanks, but not really. The buffer in which we collect is very important. RIPA buffer is no good and same with any buffer with strong detergents. I cannot snap freeze my sample. I am trying to find a good method right from the collection stage to a final stage where I could run an ELISA. Mnay thanks

#6 bob1

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Posted 20 September 2012 - 01:37 PM

In that case, you already know that some detergents don't work - so you should be able to make up a buffer that doesn't include those detergents. Some (many) ELISAs will work just fine in RIPA, but it depends on the antibody and the strength of the interaction between proteins. If you need to access the nuclear compartment you may need to think about homogenization with a Dounce homogenizer or something similar.

Starting place place of these sorts of solutions is 150 mM NaCl and 1-10 mM tris (pH 7.5), anything you add from there deviates from physiologic conditions...




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