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Protein extraction from rat aorta - (May/14/2009 )

Would anybody have an available protocol for extracting total protein from rat aorta tissue or something similar. I have used T-Per rgt from Pierce, inclusive of inhibitors, but to no avail. I am finding the tissue very difficult to homogenise using a motorised pestle and mortar and I'm wondering if anybody has any tips. Is there some kind of treatment that i can use before I homogenise in my reagent.
I have also tried homogenising over liquid nitrogen but again my protein yields are still very low. The end product will be used for Western blotting.

Many thanks

A very frustrated final yr PhD student :D

-kellyly-

I had similar issue with some collagenous tissue so what I did, I ran the tissue through freeze and thaw cycles 3 times, i.e. put an eppendorf tube in liquid nitrogen to flash freeze and then thaw it in 37C water bath. Repeat the cycle twice more. This helped to make the tissue brittle enough to crush.

I am not too sure if homogenizing over liquid nitrogen is same as this.

(P.S. - Even if you are frustrated, don't think much about it, concentrate on your work. I am in a similar boat so just a friendly suggestion. Good Luck)

-noelmathur-

noelmathur on May 14 2009, 05:39 AM said:

I had similar issue with some collagenous tissue so what I did, I ran the tissue through freeze and thaw cycles 3 times, i.e. put an eppendorf tube in liquid nitrogen to flash freeze and then thaw it in 37C water bath. Repeat the cycle twice more. This helped to make the tissue brittle enough to crush.

I am not too sure if homogenizing over liquid nitrogen is same as this.

(P.S. - Even if you are frustrated, don't think much about it, concentrate on your work. I am in a similar boat so just a friendly suggestion. Good Luck)



Thanks for that, I'll give it a go and hope for the best.

-kellyly-

Hi, I have similar problem before also. now, i using RIPA buffer for protein extraction in 5mg tissue, the total protein concentration detected is increased to 5 mg/ml compared to my previous method. But the problems is although i have tried to run the SDS PAGE and western blot, the band detected is not very clear and visualize, my target protein just appears as my other unspecific binding. I wonder what is the problems now. Isnt necessary to centrifuge the protein in 4 degree Celsius. would it effect my proteins?

-JEE-