Hi RG community,
I want to perform Western Blot on murine adipose tissue. I am currently struggling with some issues to get good protein samples. I usually have been isolating adipose tissue straight from the mice, then minced the tissue with beads or gentleMACS disrupter in protein extraction buffer containing 1% Triton-X. However, I always have contamination of red blood cells which smears my bands and make equal loading impossible. Another issue is fat contamination in the protein sample which may introduce more smearing to the Western Blot.
I am sure some adipose experts out there could help me with some good protocols. Will I need to perfuse the mice to get a good WB sample? Do I need specific buffers?
I look forward to your expertise.
Hi dear Felix, I understand how you fell about extracting protein from adipose tissues for western blot analysis for your study.
The traditional western blot is one of the most widely used analytical techniques to quantitate-specific proteins in tissues and is considered the ‘gold standard’. Recently, a novel capillary electrophoresis-based western blot technology has been developed by ProteinSimple Inc. This capillary western blot assay incorporates sample separation performed in glass capillaries and immobilization of the proteins directly onto the capillary walls, followed by immuno-probing and chemiluminescent detection.
Adipose tissue from needle biopsies will be washed with saline, frozen in liquid nitrogen, and stored at −80 °C. For these assays, tissue will be weighed and placed in a tube containing Omni ceramic homogenization beads. Tissue will be homogenized in SHBP (20 mm Tris-HCl, 1 mm EDTA, 255 mm sucrose, PH 7.4, and anti-protease cocktail tablet), 1-4 μl mg−1 tissue at 4 °C using an Omni Bead Ruptor (speed=2.10, cycles=2, time=15 s, and delay=10 s). If the extract was prepared for testing pErk1/2 or pAkt, Halt phosphatase inhibitor cocktail will be added to the homogenization buffer. The homogenate will be centrifuged at 1000 g for 10 min at 4 °C and the subnatant (whole-tissue extract) below the lipid cake will be aspirated into a new tube. Total protein concentrations of the whole-tissue extracts will be measured using Pierce BCA Protein Quantitation kit and the samples will be stored at −80 °C until assayed.
You can lyse RBCs with RBC lyzing buffer which contains ammonium chloride. Use liquid nitrogen and zirconia beads for lysis.
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