AKT1 Kit ELISA

N° du produit ABIN8071257

Ce kit ELISA Colorimetric est destiné à la mesure quantitative de Humain, Souris et Rat AKT1.

Aperçu rapide pour AKT1 Kit ELISA (ABIN8071257)

Antigène: AKT1 (V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1))

Épitope: S-nitrosylated

Reactivité: Humain, Souris, Rat

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Type d'échantillon: Cell Lysate, Tissue Lysate

Détail du produit AKT1 Kit ELISA

Fonction: S-Nitrosylated AKT ELISA Kit uses a modified 'biotin-switch' method to allow for the direct visualization of human, rat, and mouse S-Nitrosylated AKT by ELISA application.

Analytical Method: Quantitative

Ingrédients:

Size / Qty
• Anti-Pan-AKT Microplate
• 96 wells
• Positive Control
• HRP-Streptavidin
• 200 μL
• Wash Buffer
• Assay Diluent B
• Lysis Buffer
• TMB One-Step Substrate Reagent
• Stop Solution
• S-Nitrosylation Buffer A
• S-Nitrosylation Buffer B
• S-Nitrosylation Buffer C
• S-Nitrosylation Buffer D
• S-Nitrosylation Buffer E
• S-Nitrosylation Blocking Reagent
• S-Nitrosylation Reduction Reagent I
• S-Nitrosylation Reduction Reagent II
• S-Nitrosylation Labeling Reagent

Matériel non inclus:

• Acetone (pre-chilled (-20 °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

Information d'application

Indications d'application: Optimal working dilution should be determined by the investigator.

Plaque: Pre-coated

Protocole:

1. Prepare all reagents, samples and Positive Control as instructed.
2. Block unmodified free cysteines using prepared S-Nitrosylation Blocking Buffer at 50 °C with gentle rocking.
3. Precipitate proteins with ice-cold acetone.
4. Wash protein pellet twice with cold acetone/water mixture.
5. Resuspend protein pellets in Buffer containing Reducing and Labeling Reagents.
6. Precipitate proteins with ice-cold acetone.
7. Resuspend protein pellets.
8. 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.
9. Add 100 μL prepared HRP-Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
10. Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
11. Add 50 μL Stop Solution to each well. Read at 450 nm immediately.

Restrictions: For Research Use only

Stockage

Stock: -20 °C

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

Détail du antigène

Antigène: AKT1 (V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1))

Autre désignation: AKT

Sujet:

Gene Symbols: AKT1, PKB, RAC

Background: The S-Nitrosylated AKT ELISA Kit uses a modified "biotin-switch" method to allow for the direct visualization of human and mouse S-Nitrosylated AKT by ELISA. In this S-Nitrosylation biotin switch assay, unmodified free cysteines are first blocked. S-Nitrosylated cysteines are then selectively reduced for specific labeling with biotin-maleimide reagents, which irreversibly bind to the cysteine thiol that was S-Nitrosylated. Biotinylation of the newly formed thiol groups can then be detected by ELISA. An anti-pan AKT antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells and AKT 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 S-Nitrosylated AKT. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of S-Nitrosylated AKT 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): RAC-alpha serine/threonine-protein kinase (EC 2.7.11.1) (Protein kinase B) (PKB) (Protein kinase B alpha) (PKB alpha) (Proto-oncogene c-Akt) (RAC-PK-alpha)

ID gène: 207

UniProt: P31749

Pathways: Signalisation PI3K-Akt, Signalisation RTK, TCR Signaling, AMPK Signaling, Interferon-gamma Pathway, Signalisation TLR, Fc-epsilon Receptor Signaling Pathway, EGFR Signaling Pathway, Neurotrophin Signaling Pathway, Response to Water Deprivation, Regulation of Actin Filament Polymerization, Carbohydrate Homeostasis, Glycosaminoglycan Metabolic Process, Cellular Glucan Metabolic Process, Regulation of Muscle Cell Differentiation, Cell-Cell Junction Organization, Regulation of Cell Size, Skeletal Muscle Fiber Development, Regulation of Carbohydrate Metabolic Process, Hepatitis C, Protein targeting to Nucleus, CXCR4-mediated Signaling Events, Signaling Events mediated by VEGFR1 and VEGFR2, Negative Regulation of intrinsic apoptotic Signaling, Thromboxane A2 Receptor Signaling, Signaling of Hepatocyte Growth Factor Receptor, Positive Regulation of fat Cell Differentiation, VEGFR1 Specific Signals, VEGF Signaling, L'effet Warburg