Subject Area:	Biochemistry, Genetics and Molecular Biology
More specific subject area:	Adipose tissue, protein extraction, Western blot
Protocol name:	Removal of Excess Lipids (RELi) protocol for protein extraction from white, brown and beige adipose tissues.
Reagents/tools:	Tissue Collection •Dry ice •Tweezer #5, stainless steel, 11 cm long (Kent scientific corporation, catalog # INS600095) •Dissecting scissors, straight, 10 cm long (Kent Scientific Corporation, catalog # INS600393) •Iris forceps, serrated, 10 cm long (Kent Scientific Corporation, catalog # INS650915) •60 mm dish (Thermo Scientific, catalog #130181) •2.0 mL capped tubes (1 per tissue to collect) •Ethanol (EtOH) 70% Tissue Lysis •Analytical balance •Fastprep FP120 (SAVANT Instrument Inc) •1.5 mL capped tubes (4 per tissue to extract) •Ice •Carbon Steel Surgical Blade (Bard-Parker, catalog #311121) •RIPA Buffer (10x) (Cell Signaling Technologies, catalog #9806S) •Distilled water •Protease inhibitor cocktail (Sigma-Aldrich, catalog #P8340) •Phosphatase inhibitor cocktail (Sigma-Aldrich, catalog #P5726) Centrifugation and removal of excess lipids •0.1–10 μl, 2–20 μl, 20–200 μl, and 200–1000 μl micropipettes •0.1–10 μl, 2–20 μl, 20–200 μl, 200–1000 μl pipette tips •Cooling centrifuge Protein quantification and sample preparation •TECAN Infinite M1000 Pro (ThermoFisher Scientific) •Microtest plate 96 well (Sarstedt AG & Co, catalog #82.1581) •BCA kit (Sigma-Aldrich, catalog # QBCA-1KT) •Protein Standard (Sigma-Aldrich, catalog #PO914-10AMP) •Acrylamide gels 12.5% •Laemmli buffer 4X •Molecular weight precision plus protein standards (BIO-RAD, catalog #161-0375) •KimTech Science (Kimberly-Clark Professional) •PVDF membranes for protein blotting (BIO-RAD, catalog #1620177) •Ponceau S solution (Sigma-Aldrich, catalog #P7170-1L) •β -Actin (8H10D10) mouse monoclonal antibody (Cell Signaling Technologies, catalog #3700) •Bcl2 (EPR17509) rabbit monoclonal antibody (Abcam, catalog # ab182858) •eNOS mouse monoclonal antibody (BD Biosciences, catalog # 610296) •Perilipin A rabbit polyclonal antibody (Abcam, catalog # ab3526) •Histone H3 (C-16) goat polyclonal antibody (Santa-Cruz, catalog #sc-8654) •Goat Anti-Mouse IgG (H + L)-HRP Conjugate (BIO-RAD, catalog #1706516) •Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (BIO-RAD, catalog #1721019) •Donkey Anti-Goat IgG (H&L)-HRP (Abcam, catalog #ab97110) •Goat Anti-Mouse IgG (H + L)-HRP Conjugate (BIO-RAD, catalog #1706516) •Clarity Western ECL Substrate (BIO-RAD, catalog #170-5061) •ImageQuant LAS 4000 (GE Healthcare) •ImageJ image processing program (U.S. National Institutes of Health)
Experimental design:	White, brown and beige adipose tissues of C57BL/6 J male mice were collected, and protein was extracted by either the Cell Signaling Technologies (CST) method or by our novel Removal of Excess Lipids (RELi) method. Whole cell lysate proteins were quantified, run by means of Western blot and probed for β-actin levels.
Trial registration:	Not applicable
Ethics:	All experimental procedures were approved by the Animal Care Committee of the Maisonneuve-Rosemont Hospital Research Center and in accordance to the guidelines of the Canadian Council on Animal Care.
Value of the Protocol	•The optimization steps presented in this protocol provide a methodological approach to remove excess contaminating lipids and detergents during protein extraction from adipose tissue. This approach increases the quality and quantity of total extracted proteins from adipose tissue. •This improved protocol permits for proper and reproducible loading of investigated proteins and housekeeping genes in western blot analysis