[[ Category: Molecular Biology ]]
[[ Category: EMSA ]]
[[ Category: ChIP ]]
[[ Category: Subcellular Localization ]]
From [http://dx.doi.org/10.1038/ncb3080 Kang et al]
Epididymal adipose tissue was collected from chow==Materials==* Mice* Dissection Tools* 2 mL Dounce Homogenizer* Hypotonic Lysis Buffe, cool on ice: {| class="wikitable"! style="font- (nweight: bold;" | Reagent! style=15) and high"text-fatalign: center; font-fed weight: bold;" | Amount (n=750 mL) C57BL/6 mice. Pooled fat pads were minced and dounce homogenized with 10 strokes in hypotonic lysis buffer (10mM ! style="text-align: center; font-weight: bold;" | Stock Concentration! style="text-align: center; font-weight: bold;" | Final Concentration|-| HEPES, pH 7.5, 10mM KCl, | style="text-align: center;" | 500 uL| style="text-align: center;" | 1M| style="text-align: center;" | 10 mM|-| Potassium chloride| style="text-align: center;" | 500 uL| style="text-align: center;" | 1M| style="text-align: center;" | 10 mM|-| Magnesium chloride| style="text-align: center;" | 75 uL| style="text-align: center;" | 1M| style="text-align: center;" | 1.5mM MgCl2 , 250mM sucrose, 5 mM|-| Sucrose| style="text-align: center;" | 4.28g| style="text-align: center;" | Solid| style="text-align: center;" | 250 mM|-| NP40| style="text-align: center;" | 2.5 mL| style="text-align: center;" | 10%| style="text-align: center;" | 0.5% NP40|} ==Protocol==* Dissect adipose tissue into weigh boat, and protease inhibitor cocktail). Lysates were filtered through determine the weight* Mince to small <1mm pieces with scissors* Transfer to a 100 µm cell strainer clean tube and spun add one volume of '''Hypotonic Lysis Buffer''' on ice* Homogenize with 10 strokes in a 2 mL homogenizer and transfer back to an eppendorf tube* Centrifuge at 1, 500g 4C for 5 min. Lipid and cytoplasmic fractions were removed and minutes at 1500g* Carefully remove the fat pad with tweezers, store if needed* Collect supernatant into fresh tube, carefully remove all remaining cytosol* Re-suspend nuclear pellet was resuspended in 1/10 of the original lysis buffer* Quantify protein content in both nuclei and cytosol by [[ Bradford Assay ]]* Take 20-50 uL and boil in SDS-PAGE lysis buffer, store remaining cytosol or nuclear lysate in -20 or -80
==References==
Kang S, Tsai LT, Zhou Y, Evertts A, Xu S, Griffin MJ, Issner R, Whitton HJ, Garcia BA, Epstein CB, Mikkelsen TS, Rosen ED. Identification of nuclear hormone receptor pathways causing insulin resistance by transcriptional and epigenomic analysis. Nat Cell Biol. 2015; 17: 44–56. doi: [http://dx.doi.org/10.1038/ncb3080 10.1038/ncb3080].
