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Typical phospho- GSK-3β Standard Curve
50 μL of progressive 2 fold dilutions of the phosphorylated GSK-3β (Ser9) standard was run as des...
Phospho-GSK-3β (Ser9) STAR ELISA Kit
Description:
Phospho-GSK-3β (Ser9) STAR ELISA Kit
Trade Name:
Upstate (Millipore)
Qty/Pk:
96 assays
Background Information:
I. TEST PRINCIPLE The UPSTATE® colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The GSK-3β plate is coated with a specific mouse monoclonal GSK-3&beta capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing GSK-3β antigen to be captured in the plate wells. The plate is then washed to remove any non-bound unspecific material. The wells are then incubated with a specific rabbit anti-GSK-3β antibody to detect the captured phosphor-GSK-3β (Ser9) on the plate well. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard line of detection.
II. GSK-3β BACKGROUND Glycogen Synthase Kinase-3 differs from most serine/threonine kinases in that it is active in the absence of the action of signaling pathways. When it becomes phosphorylated, the protein becomes inactive. There are two isoforms that exist, GSK-3α and GSK-3β. The function of GSK-3 is to phosphorylate Glycogen Synthase and thereby inactivate it. Insulin action stimulates the PI3 Kinase pathway that results in the activation of Akt. Akt phosphorylates GSK-3, thereby inactivating it. Glycogen Synthase, GSK-3's downstream substrate, is then rapidly dephosphorylated and activated. Other GSK-3 substrates include Jun (on inhibitory sites), and eIF2B. Detection of GSK-3's phosphorylation status of the Akt site (Ser21) on GSK-3 is suitable for surrogate assays of the activation state of the pathway.
II. GSK-3β BACKGROUND Glycogen Synthase Kinase-3 differs from most serine/threonine kinases in that it is active in the absence of the action of signaling pathways. When it becomes phosphorylated, the protein becomes inactive. There are two isoforms that exist, GSK-3α and GSK-3β. The function of GSK-3 is to phosphorylate Glycogen Synthase and thereby inactivate it. Insulin action stimulates the PI3 Kinase pathway that results in the activation of Akt. Akt phosphorylates GSK-3, thereby inactivating it. Glycogen Synthase, GSK-3's downstream substrate, is then rapidly dephosphorylated and activated. Other GSK-3 substrates include Jun (on inhibitory sites), and eIF2B. Detection of GSK-3's phosphorylation status of the Akt site (Ser21) on GSK-3 is suitable for surrogate assays of the activation state of the pathway.
Key Applications:
ELISA
Species Reactivity:
- Human
- Mouse
- Rat
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
Glycogen synthase kinase-3 (GSK3) is a proline-directed serine-threonine kinase that was initially identified as a phosphorylating and inactivating glycogen synthase. Two isoforms, alpha (GSK3A; MIM 606784) and beta, show a high degree of amino acid homology (Stambolic and Woodgett, 1994 [PubMed 7980435]). GSK3B is involved in energy metabolism, neuronal cell development, and body pattern formation (Plyte et al., 1992 [PubMed 1333807]).[supplied by OMIM]
UniProt Summary:
FUNCTION: SwissProt: P49841 # Participates in the Wnt signaling pathway. Implicated in the hormonal control of several regulatory proteins including glycogen synthase, MYB and the transcription factor JUN. Phosphorylates JUN at sites proximal to its DNA-binding domain, thereby reducing its affinity for DNA.
SIZE: 420 amino acids; 46744 Da
SUBUNIT: Monomer (By similarity). Interacts with NIN and CABYR.
TISSUE SPECIFICITY: Expressed in testis, thymus, prostate and ovary and weakly expressed in lung, brain and kidney.
PTM: Phosphorylated by AKT1 and ILK1.
SIMILARITY: SwissProt: P49841 ## Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. GSK-3 subfamily. & Contains 1 protein kinase domain.
SIZE: 420 amino acids; 46744 Da
SUBUNIT: Monomer (By similarity). Interacts with NIN and CABYR.
TISSUE SPECIFICITY: Expressed in testis, thymus, prostate and ovary and weakly expressed in lung, brain and kidney.
PTM: Phosphorylated by AKT1 and ILK1.
SIMILARITY: SwissProt: P49841 ## Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. GSK-3 subfamily. & Contains 1 protein kinase domain.
Brand Family:
Upstate
Sensitivity:
Sensitivity: 1.6 Units/mL
Range of Detection: 1.6 to 100 Units/mL
Range of Detection: 1.6 to 100 Units/mL
Storage Conditions:
Maintain the unopened kit at 2-8°C until expiration date.
Precautions
• The instructions provided have been designed to optimize the kit's performance. Deviation from the instructions may result in suboptimal performance of the kit and the failure to produce accurate data.
• Caustic Material: Stop Solution. Caution: Eye, hand, face, and clothing protection should be worn when handling this material.
• Safety Warnings and Precautions: This kit is designed for research use only and not recommended for internal use in humans or animals. All chemicals should be considered potentially hazardous and principles of good laboratory practice should be followed.
• The Detection Antibody and ELISA Diluent contain sodium azide. Sodium azide may react with copper and lead plumbing to form highly explosive metal azides. Upon disposal, flush with large amounts of water to prevent azide build-up. Avoid contact with skin.
• The Anti-Rabbit IgG HRP Conjugate and HRP Diluent contain thimerosal. Thimerosal is highly toxic by inhalation, contact with skin or if swallowed. Thimerosal is a possible mutagen and should be handled accordingly.
Precautions
• The instructions provided have been designed to optimize the kit's performance. Deviation from the instructions may result in suboptimal performance of the kit and the failure to produce accurate data.
• Caustic Material: Stop Solution. Caution: Eye, hand, face, and clothing protection should be worn when handling this material.
• Safety Warnings and Precautions: This kit is designed for research use only and not recommended for internal use in humans or animals. All chemicals should be considered potentially hazardous and principles of good laboratory practice should be followed.
• The Detection Antibody and ELISA Diluent contain sodium azide. Sodium azide may react with copper and lead plumbing to form highly explosive metal azides. Upon disposal, flush with large amounts of water to prevent azide build-up. Avoid contact with skin.
• The Anti-Rabbit IgG HRP Conjugate and HRP Diluent contain thimerosal. Thimerosal is highly toxic by inhalation, contact with skin or if swallowed. Thimerosal is a possible mutagen and should be handled accordingly.
Modifications:
Phosphorylation
Gene Symbol:
GSK3B
Product Name:
Phospho-GSK-3β (Ser9) STAR ELISA Kit
Alternate Names:
glycogen synthase kinase 3 beta
Materials Required but Not Delivered:
1. Multi-channel or repeating pipettes
2. Plate shaker (optional)
3. Pipettors and tips capable of accurately measuring 1-1000 µL
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water
2. Plate shaker (optional)
3. Pipettors and tips capable of accurately measuring 1-1000 µL
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water
Analytes Available:
GSK-3β
Quality Assurance:
Routinely evaluated by ELISA.
Components:
- GSK-3β Capture Plate: (Part No. 17-472A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
- Anti-phospho-GSK-3β (Ser9) detection antibody: (Part No. 17-472B) One bottle (6 mL) of anti-phospho-GSK-3β (Ser9) detection antibody containing sodium azide, ready to use.
- ELISA Diluent: (Part No. 17-472C) One bottle (25 mL) of ELISA Diluent sodium azide, ready to use
- 25X ELISA Wash Buffer: (Part No. 17-472D) One bottle (50 mL) of 25X ELISA Wash Buffer.
- Anti-Rabbit IgG HRP conjugate: (Part No. 17-472E) One vial (125 μL) of 100X anti-rabbit HRP conjugate.
- HRP Diluent: (Part No. 17-472F) One bottle (25 mL) of HRP diluent.
- TMB Solution: (Part No. 17-472G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
- Stop Solution: (Part No. 17-472H) One bottle (25 mL) of stop solution, ready to use.
- Phosphorylated GSK-3β (Ser9) Standard: (Part No. 17-472I) Two vials of phosphorylated GSK-3β (Ser9) standard, lyophilized.
- Plate Covers: Two plate covers.
UniProt Number:
Detection Methods:
Colorimetric
Entrez Gene Number: