Difference between revisions of "Adipose Tissue Nuclear Isolation"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (copied initial protocol from Kang) |
Davebridges (Talk | contribs) (Updated protocol with more details) |
||
| Line 1: | Line 1: | ||
| + | [[ Category: Molecular Biology ]] | ||
| + | [[ Category: EMSA ]] | ||
| + | [[ Category: ChIP ]] | ||
| + | [[ Category: Subcellular Localization ]] | ||
| + | |||
From [http://dx.doi.org/10.1038/ncb3080 Kang et al] | From [http://dx.doi.org/10.1038/ncb3080 Kang et al] | ||
| − | + | ==Materials== | |
| + | * Mice | ||
| + | * Dissection Tools | ||
| + | * 2 mL Dounce Homogenizer | ||
| + | * Hypotonic Lysis Buffe, cool on ice: | ||
| + | |||
| + | {| class="wikitable" | ||
| + | ! style="font-weight: bold;" | Reagent | ||
| + | ! style="text-align: center; font-weight: bold;" | Amount (50 mL) | ||
| + | ! style="text-align: center; font-weight: bold;" | Stock Concentration | ||
| + | ! style="text-align: center; font-weight: bold;" | Final Concentration | ||
| + | |- | ||
| + | | HEPES, pH 7.5 | ||
| + | | 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;" | 1.5 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% | ||
| + | |} | ||
| + | |||
| + | ==Protocol== | ||
| + | * Dissect adipose tissue into weigh boat, and determine the weight | ||
| + | * Mince to small <1mm pieces with scissors | ||
| + | * Transfer to a clean tube and 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 4C for 5 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 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== | ==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]. | 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]. | ||
Latest revision as of 11:56, 28 March 2017
From Kang et al
Materials
- Mice
- Dissection Tools
- 2 mL Dounce Homogenizer
- Hypotonic Lysis Buffe, cool on ice:
| Reagent | Amount (50 mL) | Stock Concentration | Final Concentration |
|---|---|---|---|
| HEPES, pH 7.5 | 500 uL | 1M | 10 mM |
| Potassium chloride | 500 uL | 1M | 10 mM |
| Magnesium chloride | 75 uL | 1M | 1.5 mM |
| Sucrose | 4.28g | Solid | 250 mM |
| NP40 | 2.5 mL | 10% | 0.5% |
Protocol
- Dissect adipose tissue into weigh boat, and determine the weight
- Mince to small <1mm pieces with scissors
- Transfer to a clean tube and 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 4C for 5 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 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: 10.1038/ncb3080.
