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Chk1 (Phospho-Ser280) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01506
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Cycle
Reactivity:
Human
Detection Method:
Colorimetric
€599
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Description

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Chk1 (Phospho-Ser280)Colorimetric Cell-Based ELISA Kit

The CHK1 Phospho-Ser280 Colorimetric Cell-based ELISA Kit is a cutting-edge assay designed for the precise detection of phosphorylated CHK1 at serine 280 in cell samples. This kit offers exceptional sensitivity and specificity, ensuring dependable and consistent results for a variety of research applications.CHK1 is a key regulator of the cell cycle and DNA damage response pathway, playing a critical role in maintaining genomic stability and cell survival. Phosphorylation of CHK1 at serine 280 is a crucial post-translational modification that can impact CHK1 function and signaling pathways.

Researchers studying cell cycle checkpoints, DNA damage response, and cancer biology will find this kit invaluable for their experiments. By accurately measuring CHK1 phosphorylation at serine 280, scientists can gain insights into the molecular mechanisms governing cell cycle regulation and DNA repair processes.Overall, the CHK1 Phospho-Ser280 Colorimetric Cell-based ELISA Kit is a powerful tool for investigating CHK1 signaling dynamics and identifying potential therapeutic targets for cancer treatment and other related diseases.

Product Name:Chk1 (Phospho-Ser280) Colorimetric Cell-Based ELISA
Product Code:CBCAB01506
ELISA Type:Cell-Based
Target:Chk1 (Phospho-Ser280)
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Chk1 (Phospho-Ser280) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Chk1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Chk1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Chk1 phosphorylation.

Qualitative determination of Chk1 (Phospho-Ser280) concentration is achieved by an indirect ELISA format. In essence, Chk1 (Phospho-Ser280) is captured by Chk1 (Phospho-Ser280)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 1111, UniProt ID: O14757, OMIM: 603078, Unigene: Hs.24529
Gene Symbol:CHK1
Sub Type:Phospho
UniProt Protein Function:Serine/threonine-protein kinase which is required for checkpoint-mediated cell cycle arrest and activation of DNA repair in response to the presence of DNA damage or unreplicated DNA. May also negatively regulate cell cycle progression during unperturbed cell cycles. This regulation is achieved by a number of mechanisms that together help to preserve the integrity of the genome. Recognizes the substrate consensus sequence [R-X-X-S/T]. Binds to and phosphorylates CDC25A, CDC25B and CDC25C. Phosphorylation of CDC25A at 'Ser-178' and 'Thr-507' and phosphorylation of CDC25C at 'Ser-216' creates binding sites for 14-3-3 proteins which inhibit CDC25A and CDC25C. Phosphorylation of CDC25A at 'Ser-76', 'Ser-124', 'Ser-178', 'Ser-279' and 'Ser-293' promotes proteolysis of CDC25A. Phosphorylation of CDC25A at 'Ser-76' primes the protein for subsequent phosphorylation at 'Ser-79', 'Ser-82' and 'Ser-88' by NEK11, which is required for polyubiquitination and degradation of CDCD25A. Inhibition of CDC25 leads to increased inhibitory tyrosine phosphorylation of CDK-cyclin complexes and blocks cell cycle progression. Also phosphorylates NEK6. Binds to and phosphorylates RAD51 at 'Thr-309', which promotes the release of RAD51 from BRCA2 and enhances the association of RAD51 with chromatin, thereby promoting DNA repair by homologous recombination. Phosphorylates multiple sites within the C-terminus of TP53, which promotes activation of TP53 by acetylation and promotes cell cycle arrest and suppression of cellular proliferation. Also promotes repair of DNA cross-links through phosphorylation of FANCE. Binds to and phosphorylates TLK1 at 'Ser-743', which prevents the TLK1-dependent phosphorylation of the chromatin assembly factor ASF1A. This may enhance chromatin assembly both in the presence or absence of DNA damage. May also play a role in replication fork maintenance through regulation of PCNA. May regulate the transcription of genes that regulate cell-cycle progression through the phosphorylation of histones. Phosphorylates histone H3.1 (to form H3T11ph), which leads to epigenetic inhibition of a subset of genes. May also phosphorylate RB1 to promote its interaction with the E2F family of transcription factors and subsequent cell cycle arrest.
NCBI Summary:The protein encoded by this gene belongs to the Ser/Thr protein kinase family. It is required for checkpoint mediated cell cycle arrest in response to DNA damage or the presence of unreplicated DNA. This protein acts to integrate signals from ATM and ATR, two cell cycle proteins involved in DNA damage responses, that also associate with chromatin in meiotic prophase I. Phosphorylation of CDC25A protein phosphatase by this protein is required for cells to delay cell cycle progression in response to double-strand DNA breaks. Several alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Oct 2011]
UniProt Code:O14757
NCBI GenInfo Identifier:317373343
NCBI Gene ID:1111
NCBI Accession:O14757.2
UniProt Secondary Accession:O14757,A8K934, B4DDD0, B4DSK3, B5BTY6, F5H7S4, H2BI51
UniProt Related Accession:O14757
Molecular Weight:50,415 Da
NCBI Full Name:Serine/threonine-protein kinase Chk1
NCBI Synonym Full Names:checkpoint kinase 1
NCBI Official Symbol:CHEK1  
NCBI Official Synonym Symbols:CHK1  
NCBI Protein Information:serine/threonine-protein kinase Chk1
UniProt Protein Name:Serine/threonine-protein kinase Chk1
UniProt Synonym Protein Names:CHK1 checkpoint homolog; Cell cycle checkpoint kinase; Checkpoint kinase-1
UniProt Gene Name:CHEK1  
UniProt Entry Name:CHK1_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-Chk1 (Phospho-Ser280) Antibody, Anti-Chk1 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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