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MCM4 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00328
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Cycle
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheet

MCM4 Colorimetric Cell-Based ELISA Kit

The MCM4 Colorimetric Cell-Based ELISA Kit from Assay Genie is a powerful tool for the detection of MCM4 levels in cell samples. This kit offers high sensitivity and specificity, allowing for accurate and reliable results in a variety of research applications.MCM4 is a key protein involved in DNA replication and cell cycle regulation, making it essential for cell growth and proliferation. Dysregulation of MCM4 has been linked to various diseases, including cancer and developmental disorders, highlighting its importance as a biomarker for studying these conditions.

With the MCM4 Colorimetric Cell-Based ELISA Kit, researchers can easily and efficiently measure MCM4 levels in cell cultures, providing valuable insights into cellular processes and potential therapeutic targets. Trust Assay Genie for innovative solutions in cell-based research.

Product Name:MCM4 Colorimetric Cell-Based ELISA
Product Code:CBCAB00328
ELISA Type:Cell-Based
Target:MCM4
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The MCM4 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MCM4 protein expression profile in cells. The kit can be used for measuring the relative amounts of MCM4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MCM4.

Qualitative determination of MCM4 concentration is achieved by an indirect ELISA format. In essence, MCM4 is captured by MCM4-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: 4173, UniProt ID: P33991, OMIM: 602638, Unigene: Hs.460184
Gene Symbol:MCM4
Sub Type:None
UniProt Protein Function:MCM4: a mini-chromosome maintenance protein, essential for the initiation of eukaryotic genome replication. The MCM hexameric protein complex is a key component of the pre-replication complex. The complex consisting of this protein and MCM2, 6 and 7 possesses DNA helicase activity. Phosphorylation by CDC2 reduces the DNA helicase activity and chromatin binding of the complex.
UniProt Protein Details:

Protein type:EC 3.6.4.12; DNA replication

Chromosomal Location of Human Ortholog: 8q11.2

Cellular Component: MCM complex; membrane; nuclear chromosome, telomeric region; nucleoplasm

Molecular Function:ATP-dependent DNA helicase activity; protein binding

Biological Process: DNA replication; G1/S transition of mitotic cell cycle

Disease: Natural Killer Cell And Glucocorticoid Deficiency With Dna Repair Defect

NCBI Summary:The protein encoded by this gene is one of the highly conserved mini-chromosome maintenance proteins (MCM) that are essential for the initiation of eukaryotic genome replication. The hexameric protein complex formed by MCM proteins is a key component of the pre-replication complex (pre_RC) and may be involved in the formation of replication forks and in the recruitment of other DNA replication related proteins. The MCM complex consisting of this protein and MCM2, 6 and 7 proteins possesses DNA helicase activity, and may act as a DNA unwinding enzyme. The phosphorylation of this protein by CDC2 kinase reduces the DNA helicase activity and chromatin binding of the MCM complex. This gene is mapped to a region on the chromosome 8 head-to-head next to the PRKDC/DNA-PK, a DNA-activated protein kinase involved in the repair of DNA double-strand breaks. Alternatively spliced transcript variants encoding the same protein have been reported. [provided by RefSeq, Jul 2008]
UniProt Code:P33991
NCBI GenInfo Identifier:68571766
NCBI Gene ID:4173
NCBI Accession:P33991.5
UniProt Secondary Accession:P33991,Q8NEH1, Q99658,
UniProt Related Accession:P33991
Molecular Weight:96,558 Da
NCBI Full Name:DNA replication licensing factor MCM4
NCBI Synonym Full Names:minichromosome maintenance complex component 4
NCBI Official Symbol:MCM4  
NCBI Official Synonym Symbols:NKCD; CDC21; CDC54; NKGCD; hCdc21; P1-CDC21  
NCBI Protein Information:DNA replication licensing factor MCM4
UniProt Protein Name:DNA replication licensing factor MCM4
UniProt Synonym Protein Names:CDC21 homolog; P1-CDC21
Protein Family:DNA replication licensing factor
UniProt Gene Name:MCM4  
UniProt Entry Name:MCM4_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-MCM4 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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