3 replies to this topic

[kellyly]

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

[noelmathur]

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 37°C 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)

[kellyly]

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 37°C 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.

[JEE]

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?