SIRT1 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.
SIRT1 Antibody detects endogenous levels of total SIRT1.
The sirtuin family represents a unique set of enzymes that respond directly to the energy state of the cell in regulating its intrinsic enzymatic functions. SIRT1 is most conserved to its ancestral yeast Sir2, in both protein structure and function, and acts solely as a deacetylase, similar to SIRT2, while SIRT4 and SIRT6 do not have strong deacetylase activity. SIRT1 has been comprehensively studied in higher organisms, and the focus has been on the substrates of SIRT1 deacetylation. Besides the ability of SIRT1 to deacetylate H1, H3, and H4, SIRT1 is also found to deacetylate a critical master regulator in p53, suggesting a role in tumor suppression. Since the initial discovery of SIRT1 deacetylation of p53, many diverse protein substrates have been identified, including FOXO3a, PGC-1α, and PPARγ. These SIRT1 substrates have linked the metabolic state of the cell to various diseases, including cancer, diabetes, and neurodegenerative diseases.