SIRT6 Rabbit Recombinant mAb
- 1. Why choose rabbit monoclonal antibody?
- Rabbit monoclonal antibody has over 100 times higher affinity than that of mouse monoclonal.
- 2. Why choose recombinant antibodies?
- Recombinant antibodies are known for higher purity and minimal deviation between batches, versus regular antibodies.
|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
SIRT6 Antibody detects endogenous levels of SIRT6.
Sirtuins are the mammalian homologs of the yeast histone deacetylase Sir2. Among members of this family, SIRT6 appears to have particular significance in regulating metabolism, DNA repair and lifespan. It is a major player in energy homeostasis. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. A recent research reports that SIRT6 regulates HSC homeostasis through the transcriptional repression of Wnt target genes. Mechanistically, SIRT6 inhibits the transcription of Wnt target genes by interacting with transcription factor LEF1 and deacetylating histone 3 at lysine 56. Pharmacological inhibition of the Wnt pathway rescues the aberrant proliferation and functional defect in SIRT6-deficient HSCs.