AKT1 ELISA Kit

Produktnummer ABIN8071257

Dieses Colorimetric ELISA kit dient der quantitativen Messung von Human, Maus und Ratte AKT1.

Kurzübersicht für AKT1 ELISA Kit (ABIN8071257)

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

Bindungsspezifität: S-nitrosylated

Reaktivität: Human, Maus, Ratte

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Proben: Cell Lysate, Tissue Lysate

Produktdetails AKT1 ELISA Kit

Verwendungszweck: 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.

Analytische Methode: Quantitative

Bestandteile:

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

Benötigtes Material:

• 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

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Plattentyp: Pre-coated

Protokoll:

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.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C

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

Details zu AKT1

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

Andere Bezeichnung: AKT

Hintergrund:

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)

Gen-ID: 207

UniProt: P31749

Pathways: PI3K-Akt Signalweg, RTK Signalweg, T-Zell Rezeptor Signalweg, AMPK Signaling, Interferon-gamma Pathway, TLR Signalweg, Fc-epsilon Rezeptor Signalübertragung, EGFR Signaling Pathway, Neurotrophin Signalübertragung, 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, Warburg Effekt