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Trizol and Protein extraction


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#1 peanuts

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Posted 04 September 2011 - 03:02 PM

I wonder does anyone try reserve the protein after RNA extraction with Trizol?

The protocol suggests to use guanidine-hcl after protein precipitation, I wonder is there any alternatives that I can use.

Thank you!

#2 Kamran

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Posted 04 September 2011 - 08:36 PM

Don't know alternative for Guanidine HCl but isolation protein from Trizol based method is really........difficult.

Last week I did it in my lab however, protein pellet did not dissolve completely even after heating, adding cocktail of detergents and prolong incubation at 4°C. Finally, I suspended that partially solubilized protein pellet in SDS-PAGE sample loading buffer, heated at 95°C for 3 min and used supernatant for subsequent SDS-PAGE.

It worked and i got strong signal for my membrane protein on Western Blot however, housekeeping gene expression pattern was not similar owing to unequal sample loading as i could not estimate protein concentration due to presence of high detergents concentration.

Regards,

M.K. Raja

#3 peanuts

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Posted 05 September 2011 - 06:06 AM

Thanks for your reply, Raja. I just found out that ammonium acetate can also be used after phenol extraction.

Western blot is exactly what I planed to do in addition to RNA extraction. I will see the resuspension of the protein pellet.




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