Sirtuin 1/SIRT1 Antibody

Sirtuin 1/SIRT1 Antibody:
Immunohistochemistry-paraffin embedded sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.

Staining:
1. Wash sections in dH2O three times for 5 minutes each.
2. Wash section in wash buffer (1X PBS/0.1% Tween-20 (1X PBST)) for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1X PBST, 5% goat serum) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul primary antibody diluted in 1X PBST, 5% goat serum to each section. Incubate overnight at 4C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated secondary antibody, diluted in 1X PBST, 5% goat serum. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Striptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in dH2O.
12. Counterstain sections in hematoxylin.
13. Wash sections in dH2O two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.

Sirtuin 1/SIRT1 Antibody:
Western Blot Protocol

1. Perform SDS-PAGE (4-12% MOPS) on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.
3. Rinse membrane with dH2O and then stain the blot using Ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.
4. Rinse the blot in TBS for approximately 5 minutes.
5. Block the membrane using 5% NFDM + 1% BSA in TBS + Tween, 1 hour at RT.
6. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
7. Dilute the rabbit anti-SIRT1 primary antibody (NBP1-49540) in blocking buffer and incubate 1 hour at room temperature.
8. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
9. Apply the diluted rabbit-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions (Pierce ECL).

*Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.