Difference between revisions of "HDAC Activity Sex Differences"
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| + | ==Materials== | ||
| + | EpiQuik Nuclear Extraction Kit [https://www.epigentek.com/catalog/epiquik-nuclear-extraction-kit-p-149.html Epigentek OP-0002-1] 100 Extractions $184.00 | ||
| + | Epigenase HDAC Activity/Inhibition Direct Assay Kit (Colorimetric) [https://www.epigentek.com/catalog/epigenase-hdac-activityinhibition-direct-assay-kit-colorimetric-p-2867.html Epigentek P-4034-96] 96 assays $415.00 | ||
| + | ==Tissue Nuclear Extraction== | ||
| + | ===For Tissue samples=== | ||
| + | 1. Weigh tissue and cut it into small pieces. | ||
| + | 2. Add to 2ml homogenizer tube with NE1 buffer (5000 ul/mg of tissue) | ||
| + | 3. Homogenize (50-60 strokes). | ||
| + | 4. Incubate on ice for 15mins | ||
| + | 5. Centrifuge for 10mins at 12000 rpm at 4C | ||
| + | 6. Remove the supernatant | ||
| + | ===Nuclear Extract Preparation=== | ||
| + | 1. Add 2 volumes (based on pellet size) of NE2 containing DTT and PIC to nuclear pellet (2500 ul / mg of initial tissue) | ||
| − | + | 2. Incubate the extract on ice for 15mins with vortex (5secs) every 3 mins. (The tissue extract can be further sonicated for 3 x 10 seconds to increase nuclear protein extraction). | |
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| + | 3. Centrifuge the suspension for 10mins at 14000 rpm at 4C and transfer the supernatant into a new micro-centrifuge vial. | ||
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| + | 4. Use immediately or aliquot and freeze supernatant at -80C until further use. | ||
| + | Be sure to save aliquot for protein concentration. | ||
| + | |||
| + | 5. Measure the protein concentration of the nuclear extract | ||
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| + | [[Category: Epigenetics]] | ||
Latest revision as of 11:54, 7 July 2022
Materials
EpiQuik Nuclear Extraction Kit Epigentek OP-0002-1 100 Extractions $184.00
Epigenase HDAC Activity/Inhibition Direct Assay Kit (Colorimetric) Epigentek P-4034-96 96 assays $415.00
Tissue Nuclear Extraction
For Tissue samples
1. Weigh tissue and cut it into small pieces.
2. Add to 2ml homogenizer tube with NE1 buffer (5000 ul/mg of tissue)
3. Homogenize (50-60 strokes).
4. Incubate on ice for 15mins
5. Centrifuge for 10mins at 12000 rpm at 4C
6. Remove the supernatant
Nuclear Extract Preparation
1. Add 2 volumes (based on pellet size) of NE2 containing DTT and PIC to nuclear pellet (2500 ul / mg of initial tissue)
2. Incubate the extract on ice for 15mins with vortex (5secs) every 3 mins. (The tissue extract can be further sonicated for 3 x 10 seconds to increase nuclear protein extraction).
3. Centrifuge the suspension for 10mins at 14000 rpm at 4C and transfer the supernatant into a new micro-centrifuge vial.
4. Use immediately or aliquot and freeze supernatant at -80C until further use. Be sure to save aliquot for protein concentration.
5. Measure the protein concentration of the nuclear extract