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MCM5 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00748
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Cycle
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

MCM5 Colorimetric Cell-Based ELISA Kit

The Human MCM5 Colorimetric Cell-Based ELISA Kit is a highly sensitive and specific assay designed for the accurate detection of MCM5 levels in cell lysates, serum, and plasma samples. This kit provides reliable and reproducible results, making it ideal for a variety of research applications.MCM5 is a key protein involved in DNA replication and cell proliferation, making it a valuable biomarker for studying various diseases and conditions, including cancer, genetic disorders, and developmental abnormalities.

By accurately measuring MCM5 levels, researchers can gain insight into cellular processes and potentially develop new therapeutic approaches.Overall, the Human MCM5 Colorimetric Cell-Based ELISA Kit offers researchers a powerful tool for studying DNA replication and cell cycle regulation, with the potential to contribute to advancements in biomedical research and clinical diagnostics.

Product Name:MCM5 Colorimetric Cell-Based ELISA
Product Code:CBCAB00748
ELISA Type:Cell-Based
Target:MCM5
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The MCM5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MCM5 protein expression profile in cells. The kit can be used for measuring the relative amounts of MCM5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MCM5.

Qualitative determination of MCM5 concentration is achieved by an indirect ELISA format. In essence, MCM5 is captured by MCM5-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: 4174, UniProt ID: P33992, OMIM: 602696, Unigene: Hs.517582
Gene Symbol:MCM5
Sub Type:None
UniProt Protein Function:MCM5: Acts as component of the MCM2-7 complex (MCM complex) which is the putative replicative helicase essential for 'once per cell cycle' DNA replication initiation and elongation in eukaryotic cells. The active ATPase sites in the MCM2-7 ring are formed through the interaction surfaces of two neighboring subunits such that a critical structure of a conserved arginine finger motif is provided in trans relative to the ATP-binding site of the Walker A box of the adjacent subunit. The six ATPase active sites, however, are likely to contribute differentially to the complex helicase activity. Interacts with MCMBP. Belongs to the MCM family.
UniProt Protein Details:

Protein type:Cell cycle regulation; EC 3.6.4.12

Chromosomal Location of Human Ortholog: 22q13.1

Cellular Component: nucleoplasm; MCM complex; membrane; nucleus

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

Biological Process: DNA replication initiation; DNA strand elongation during DNA replication; mitotic cell cycle; DNA replication; DNA duplex unwinding; G1/S transition of mitotic cell cycle

NCBI Summary:The protein encoded by this gene is structurally very similar to the CDC46 protein from S. cerevisiae, a protein involved in the initiation of DNA replication. The encoded protein is a member of the MCM family of chromatin-binding proteins and can interact with at least two other members of this family. The encoded protein is upregulated in the transition from the G0 to G1/S phase of the cell cycle and may actively participate in cell cycle regulation. [provided by RefSeq, Jul 2008]
UniProt Code:P33992
NCBI GenInfo Identifier:19858646
NCBI Gene ID:4174
NCBI Accession:P33992.5
UniProt Secondary Accession:P33992,O60785, Q14578, Q9BTJ4, Q9BWL8,
UniProt Related Accession:P33992
Molecular Weight:82,286 Da
NCBI Full Name:DNA replication licensing factor MCM5
NCBI Synonym Full Names:minichromosome maintenance complex component 5
NCBI Official Symbol:MCM5  
NCBI Official Synonym Symbols:CDC46; P1-CDC46  
NCBI Protein Information:DNA replication licensing factor MCM5; CDC46 homolog; minichromosome maintenance deficient 5 (cell division cycle 46); MCM5 minichromosome maintenance deficient 5, cell division cycle 46
UniProt Protein Name:DNA replication licensing factor MCM5
UniProt Synonym Protein Names:CDC46 homolog; P1-CDC46
Protein Family:Minichromosome maintenance protein
UniProt Gene Name:MCM5  
UniProt Entry Name:MCM5_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-MCM5 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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