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p70 S6 Kinase (Phospho-Thr229) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00044
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Death
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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p70 S6 Kinase (Phospho-Thr229)Colorimetric Cell-Based ELISA Kit

The p70 S6 Kinase Phospho-Thr229 Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers studying cell signaling pathways and protein phosphorylation dynamics. This kit is specifically designed for the accurate detection of phosphorylated p70 S6 Kinase at threonine 229 in cell lysates, providing valuable insights into cell growth, proliferation, and metabolism.With high sensitivity and specificity, this ELISA kit delivers reliable and reproducible results, making it suitable for a wide range of research applications. The phosphorylation of p70 S6 Kinase at threonine 229 is known to play a critical role in regulating protein synthesis and cell growth, making this kit essential for studying cellular responses to various stimuli and drug treatments.

Whether you are investigating cancer biology, metabolic disorders, or cellular responses to external signals, the p70 S6 Kinase Phospho-Thr229 Colorimetric Cell-Based ELISA Kit will provide you with the accurate and precise data you need to advance your research. Trust Assay Genie for high-quality ELISA kits that deliver exceptional performance and reliability.

Product Name:p70 S6 Kinase (Phospho-Thr229) Colorimetric Cell-Based ELISA
Product Code:CBCAB00044
ELISA Type:Cell-Based
Target:p70 S6 Kinase (Phospho-Thr229)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The p70 S6 Kinase (Phospho-Thr229) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect p70 S6 Kinase protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated p70 S6 Kinase in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on p70 S6 Kinase phosphorylation.

Qualitative determination of p70 S6 Kinase (Phospho-Thr229) concentration is achieved by an indirect ELISA format. In essence, p70 S6 Kinase (Phospho-Thr229) is captured by p70 S6 Kinase (Phospho-Thr229)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 6198, UniProt ID: P23443, OMIM: 608938, Unigene: Hs.463642
Gene Symbol:RPS6KB1
Sub Type:Phospho
UniProt Protein Function:p70S6K: an AGC kinase of the RSK family that is required for cell growth and G1 cell cycle progression. Is phosphorylated and activated by mTOR in mitogenic pathways downstream of phosphoinositide 3 kinase (PI3K). Phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs. Activity is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains. Mouse knockout shows symptoms of insulin resistance, and increased insulin senstivity, resulting in protection against diet-induced obesity. Protein expression and activation upregulated in colon adenocarcinoma cell lines. Increased expression in breast cancer correlated with poor survival. Selectively amplified and overexpressed within the 17q23 breast cancer amplicon. Two isoforms produced by alternative initiation have been reported.
UniProt Protein Details:

Protein type:EC 2.7.11.1; Translation; Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; Kinase, protein; AGC group; RSK family; p70 subfamily

Chromosomal Location of Human Ortholog: 17q23.1

NCBI Summary:This gene encodes a member of the ribosomal S6 kinase family of serine/threonine kinases. The encoded protein responds to mTOR (mammalian target of rapamycin) signaling to promote protein synthesis, cell growth, and cell proliferation. Activity of this gene has been associated with human cancer. Alternatively spliced transcript variants have been observed. The use of alternative translation start sites results in isoforms with longer or shorter N-termini which may differ in their subcellular localizations. There are two pseudogenes for this gene on chromosome 17. [provided by RefSeq, Jan 2013]
UniProt Code:P23443
NCBI GenInfo Identifier:54041234
NCBI Gene ID:6198
NCBI Accession:P23443.2
UniProt Secondary Accession:P23443,Q7Z721, B2R779, B4DLT4, B4DTG1, E7ESB8, F6UYM1
UniProt Related Accession:P23443
Molecular Weight:525
NCBI Full Name:Ribosomal protein S6 kinase beta-1
NCBI Synonym Full Names:ribosomal protein S6 kinase, 70kDa, polypeptide 1
NCBI Official Symbol:RPS6KB1  
NCBI Official Synonym Symbols:S6K; PS6K; S6K1; STK14A; p70-S6K; p70 S6KA; p70-alpha; S6K-beta-1; p70(S6K)-alpha  
NCBI Protein Information:ribosomal protein S6 kinase beta-1; p70 S6 kinase, alpha; ribosomal protein S6 kinase I; serine/threonine kinase 14 alpha; serine/threonine-protein kinase 14A
UniProt Protein Name:Ribosomal protein S6 kinase beta-1
UniProt Synonym Protein Names:70 kDa ribosomal protein S6 kinase 1; P70S6K1; p70-S6K 1; Ribosomal protein S6 kinase I; Serine/threonine-protein kinase 14A; p70 ribosomal S6 kinase alpha; p70 S6 kinase alpha; p70 S6K-alpha; p70 S6KA
Protein Family:Ribosomal protein S6 kinase
UniProt Gene Name:RPS6KB1  
UniProt Entry Name:KS6B1_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-p70 S6 Kinase (Phospho-Thr229) Antibody, Anti-p70 S6 Kinase Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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