Tissue extraction from mice organ

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

3 posts Page 1 of 1
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. :idea:
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.
3 posts Page 1 of 1

Who is online

In total there are 5 users online :: 0 registered, 0 hidden and 5 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: No registered users and 5 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry