Chromatography Forum

Hi guys,
I’m going to start new research which the samples are from mice tissue like liver, kidney, spleen, skin, bone, muscle.

Is anybody out there have experience extracting the protein from those kind of sample ? I’m quite new in this merit. My laboratory is provided with ultrasound sonicator. I plan to use him, but I’m stuck with the insoluble phase. After sonicated, I filter the supernatant (yielded from centrifugation 10000g 5min) with milipore filter, but result came out with the insoluble material in it. I need to add the Nitric acid to the supernatant and it getting turbid.
I’m not sure what to do. All I do is following what my seniors did and that is the result.
So, here I am come for a help.

This should be a rather straightforward procedure. Homogenize the tissue in some type of buffer (Tris-HCl or should work), sonicate and add some protease inhibitors, centrifuge, and your supernatant should contain the protein.

The protocol which I have used for different tissues - heart, liver and kidney -
Homogenized (~ 200 mg) in protein extraction buffer (Tris-HCl, NP-40, NaCl, EDTA, NaN3, and PMSF at pH 7.5) with freshly added protease inhibitors (DTT, leupeptin and aprotinin) using mortar- pestle or using sonication method and centrifuge lysate at 27,000g for 20 min. Collect the supernatent and determine protein concentration to
using protein assay e.g. Pierce Protein Reagent Assay BCA Kit.
We have found this method consistent and seems to work for majority of tissues.