I often extract embryonic brain lysate using

50 mM Tris-Cl pH 7.4 , 150 mM NaCl, 1% Triton Buffer + protease and phosphatase inhibitors. I then pipette up and down and spin in a tabletop centrifuge cooled to 4 degrees C at full speed for 10 minutes. This lyses both the cytoplasmic fraction and nuclear fraction.

For adult brain tissue, I first flash freeze the tissue and then use the same process as above.

I have also used RIPA buffer in the past, and this works well. Alternatively, you can use the Urea lysis buffer recipe attached below. This works well for phospho-proteins and other large sized proteins.

Good luck!

Simply use the normal sample buffer for SDS electrophoresis. If you have large amounts of grease in your sample, you can use (water saturated) n-butanol for solvent extraction. Normally you need not that much protein (20 µg/mL) is fine, so that you can use small amounts of brain in a large volume. The best is is to estimate the protein content by photometry at 280 nm. 1 Unit = 1 mg/mL.

I often extract embryonic brain lysate using

50 mM Tris-Cl pH 7.4 , 150 mM NaCl, 1% Triton Buffer + protease and phosphatase inhibitors. I then pipette up and down and spin in a tabletop centrifuge cooled to 4 degrees C at full speed for 10 minutes. This lyses both the cytoplasmic fraction and nuclear fraction.

For adult brain tissue, I first flash freeze the tissue and then use the same process as above.

I have also used RIPA buffer in the past, and this works well. Alternatively, you can use the Urea lysis buffer recipe attached below. This works well for phospho-proteins and other large sized proteins.

Good luck!

Hi,

Indeed RIPA could nicely being used for most of the purpose. If you are not sure how "soluble" is your protein of interest in a buffer you could keep the pellet to further extract it with a stronger extraction buffer. In this way you could use at fisrt rather mild extraction buffer such as NP-40% based ones and then increase stringency by adding SDS and deoxycholate (more close to RIPA buffer) and the pellet could even being further extract using chaotropic agent such as urea. For protein estimation content be conscience that some detergent absorbe at 280 thereby rending the photometric approach not applicable. You could still use some colorometric such as BCA or bradfort depending on your specific conditions.

Hope this helps

Best wishes

Dear Biliana Thanks for your detailed explanation and I willu now confidently go for RIPA buffer.

Dear Daniel ... Thanks for comments about Protein quantification. I will use BCA or Bradfort method let see how it works..

I really appreciate the contribution of all of you

Stay bless