Product Images
Typical PRAS40 Standard Curve
100 μL of progressive 2 fold dilutions of the PRAS40 standard included in the kit and run as described in ...
PRAS40 STAR ELISA Kit
Description:
PRAS40 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 PRAS40 plate is coated with a specific mouse monoclonal PRAS40 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 PRAS40 antigen to be captured in the plate wells. The plate is then washed to remove any unbound non-specific material. The wells are then incubated with a specific rabbit anti-phospho-PRAS40 (Thr246) antibody to detect the captured PRAS40 on the plate well that is phosphorylated on Thr246. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. This allows for a sensitive enzymatic detection of the sample.
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 generate a standard curve for phosphorylated PRAS40 (Thr246) measurement.
II. PRAS40 BACKGROUND
PRAS40 (Proline Rich Akt Substrate, 40kDa), also known as AKT1S1, is a ubiquitously express protein that is phosphorylated on residue Thr246 via the PI3K/Akt pathway. It was originally discovered as an Akt substrate as it has the putative Akt phosphorylation motif of RxRxxpS/pT2. This phosphorylation allows it to bind 14-3-3 and Raptor of the mTOR complex mTORC1. PRAS40 is known to bind to and regulate mTORC1 (mTOR, Raptor, mLST8) kinases activity that is activated by insulin downstream of PI3K and Akt and subsequently phosphorylates p70S6K and 4EBP1. PRAS40 is known to have a putative TOS motif (FVMDE) that allows it bind to Raptor of mTORC1 in the absence of insulin. It is thought that through it binding to the Raptor, PRAS40 inhibits the kinase activity of mTORC1 by preventing to bind to its substrates such as p70S6K and 4EBP1.
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 PRAS40 plate is coated with a specific mouse monoclonal PRAS40 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 PRAS40 antigen to be captured in the plate wells. The plate is then washed to remove any unbound non-specific material. The wells are then incubated with a specific rabbit anti-phospho-PRAS40 (Thr246) antibody to detect the captured PRAS40 on the plate well that is phosphorylated on Thr246. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. This allows for a sensitive enzymatic detection of the sample.
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 generate a standard curve for phosphorylated PRAS40 (Thr246) measurement.
II. PRAS40 BACKGROUND
PRAS40 (Proline Rich Akt Substrate, 40kDa), also known as AKT1S1, is a ubiquitously express protein that is phosphorylated on residue Thr246 via the PI3K/Akt pathway. It was originally discovered as an Akt substrate as it has the putative Akt phosphorylation motif of RxRxxpS/pT2. This phosphorylation allows it to bind 14-3-3 and Raptor of the mTOR complex mTORC1. PRAS40 is known to bind to and regulate mTORC1 (mTOR, Raptor, mLST8) kinases activity that is activated by insulin downstream of PI3K and Akt and subsequently phosphorylates p70S6K and 4EBP1. PRAS40 is known to have a putative TOS motif (FVMDE) that allows it bind to Raptor of mTORC1 in the absence of insulin. It is thought that through it binding to the Raptor, PRAS40 inhibits the kinase activity of mTORC1 by preventing to bind to its substrates such as p70S6K and 4EBP1.
Key Applications:
ELISA
Species Reactivity:
Human
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:
AKT1S1 is a proline-rich substrate of AKT (MIM 164730) that binds 14-3-3 protein (see YWHAH, MIM 113508) when phosphorylated (Kovacina et al., 2003 [PubMed 12524439]).[supplied by OMIM]
UniProt Summary:
FUNCTION: SwissProt: Q96B36 # May play an important role in phosphatidylinositol 3- kinase (PI3K)-AKT1 survival signaling. Substrate for AKT1 phosphorylation, but can also be activated by AKT1-independent mechanisms. Its role in survival signaling pathways may be modulated by oxidative stress. May also play a role in nerve growth factor-mediated neuroprotection.
SIZE: 256 amino acids; 27383 Da
SUBUNIT: The phosphorylated form interacts with 14-3-3.
SUBCELLULAR LOCATION: Cytoplasm, cytosol (By similarity). Note=Found in the cytosolic fraction of the brain (By similarity).
TISSUE SPECIFICITY: Widely expressed with highest levels of expression in liver and heart. Expressed at higher levels in cancer cell lines (e.g. A549 and HeLa) than in normal cell lines (e.g. HEK293).
SIZE: 256 amino acids; 27383 Da
SUBUNIT: The phosphorylated form interacts with 14-3-3.
SUBCELLULAR LOCATION: Cytoplasm, cytosol (By similarity). Note=Found in the cytosolic fraction of the brain (By similarity).
TISSUE SPECIFICITY: Widely expressed with highest levels of expression in liver and heart. Expressed at higher levels in cancer cell lines (e.g. A549 and HeLa) than in normal cell lines (e.g. HEK293).
Components:
- 1. Capture Plate pre-coated with anti-PRAS40 antibody: (Part No. 17-477A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
- 2. Anti-PRAS40 detection antibody: (Part No. 17-477B) One bottle (11 mL) of anti PRAS40 detection antibody containing sodium azide, ready to use.
- 3. ELISA Diluent: (Part No. 17-477C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use
- 4. 25X ELISA Wash Buffer: (Part No. 17-477D) One bottle (50 mL) of 25X ELISA Wash Buffer.
- 5. Anti-Rabbit IgG HRP conjugate: (Part No. 17-477E) One vial (125 μL) of 100X anti-rabbit HRP conjugate.
- 6. HRP Diluent: (Part No. 17-477F) One bottle (25 mL) of HRP Diluent
- 7. TMB Solution: (Part No. 17-477G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
- 8. Stop Solution: (Part No. 17-477H) One bottle (25 mL) of stop solution, ready to use.
- 9. PRAS40 Standard: (Part No. 17-477I) Two vials of PRAS40 standard, lyophilized.
- 10. Plate Covers: Two plate covers.
Brand Family:
Upstate
Sensitivity:
Sensitivity: 15.6 pg/mL
Range of Detection: 31.2 to 2000 pg/mL
Range of Detection: 31.2 to 2000 pg/mL
UniProt Number:
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.
Detection Methods:
Colorimetric
Gene Symbol:
- AKT1S1
- Lobe
- MGC2865
- PRAS40
Product Name:
PRAS40 STAR ELISA Kit
Entrez Gene Number:
Alternate Names:
Signal Transcduction Assay Reaction PRAS40 ELISA
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