SIRT1 ELISA Kit

Catalog No. ABIN8070800

Human SIRT1 ELISA Kit, Colorimetric assay for quantification of Human SIRT1.

Quick Overview for SIRT1 ELISA Kit (ABIN8070800)

Target: SIRT1 (Sirtuin 1 (SIRT1))

Binding Specificity: Oxidized Form, total

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Cell Lysate, Tissue Lysate

Product Details SIRT1 ELISA Kit

Purpose: Measures oxidated SIRT1 (Sirtuin 1) and total SIRT1 in human cell and tissue lysates, enabling precise assessment of SIRT1 oxidation by ELISA. This assay can be developed for other species (mouse, rat, etc.) upon request. Contact techsupport@raybiotech.com.

Analytical Method: Quantitative

Components:

Size / Qty
• Microplate Coated With Anti-Pan-SIRT1 Antibody
• Positive Control
• HRP-Conjugated Streptavidin
• 200 μL
• Wash Buffer
• Assay Diluent
• Lysis Buffer
• TMB One-Step Substrate Reagent
• Stop Solution
• Oxidation Buffer A
• Oxidation Buffer B
• Oxidation Labeling Reagent
• Oxidation Stabilizing Reagent

Material not included:

• 20 % (v/v) trichloroacetic acid (TCA) solution, ice-cold
• 1:1 (v/v) ethanol:ethyl acetate
• Benchtop centrifuge and microcentrifuge (4 °C)
• Microplate reader capable of measuring absorbance at 450 nm
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1 liter graduated cylinders
• Log-log graph paper or computer and software for ELISA data analysis
• Absorbent paper
• Tubes to prepare positive control or sample dilutions
• Distilled or deionized water
• Shaker

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and Positive Control as instructed.
2. Label samples with Oxidation Labeling Reagent
3. Add Oxidation Stabilizing Reagent to samples.
4. Precipitate proteins with ice-cold 20 % TCA.
5. Wash protein pellet twice with 1:1 (v/v) ethanol:ethyl acetate.
6. Resuspend protein pellets in Oxidation Buffer B.
7. Add 100 μL sample and Positive Control to each well. Incubate 2.5 hours at room temperature or overnight at 4 °C with gentle shaking.
8. Add 100 μL prepared HRP-Conjugated Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
9. Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
10. Add 50 μL Stop Solution to each well. Read at 450 nm immediately.

Restrictions: For Research Use only

Handling

Storage: -20 °C

Storage Comment: The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.

Target Details for SIRT1

Target: SIRT1 (Sirtuin 1 (SIRT1))

Alternative Name: Sirtuin 1

Background:

Gene Symbols: SIRT1, SIR2L1

Background: The Oxidated SIRT1 ELISA Kit uses the "biotin probes" method to biotin label carbonyl groups of oxidized proteins. In this method, the biotin probes first react with carbonyl groups of oxidized proteins to form unstable Schiff bases, which are then further reduced to more stable amines. The biotinylated proteins can then be detected by ELISA. An anti-pan SIRT1 antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells, and SIRT1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed, and HRP-conjugated streptavidin is used to detect oxidated SIRT1. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of oxidated SIRT1 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Protein Name (Synonyms): NAD-dependent protein deacetylase sirtuin-1 (hSIRT1), NAD-dependent protein deacylase sirtuin-1, Regulatory protein SIR2 homolog 1, SIR2-like protein 1 (hSIR2)

Gene ID: 23411

UniProt: Q96EB6

Pathways: MAPK Signaling, Intracellular Steroid Hormone Receptor Signaling Pathway, Regulation of Intracellular Steroid Hormone Receptor Signaling, Carbohydrate Homeostasis, Positive Regulation of Endopeptidase Activity, Regulation of Carbohydrate Metabolic Process, Positive Regulation of Response to DNA Damage Stimulus, Negative Regulation of intrinsic apoptotic Signaling