Cell Cycle ELISA Kits

DP-1 Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00626
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Cycle
Reactivity:
Human
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:DP-1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00626
ELISA Type:Cell-Based
Target:DP-1
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The DP-1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect DP-1 protein expression profile in cells. The kit can be used for measuring the relative amounts of DP-1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on DP-1.

Qualitative determination of DP-1 concentration is achieved by an indirect ELISA format. In essence, DP-1 is captured by DP-1-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: 7027, UniProt ID: Q14186, OMIM: 189902, Unigene: Hs.79353
Gene Symbol:TFDP1
Sub Type:None
UniProt Protein Function:TFDP1: Can stimulate E2F-dependent transcription. Binds DNA cooperatively with E2F family members through the E2 recognition site, 5'-TTTC[CG]CGC-3', found in the promoter region of a number of genes whose products are involved in cell cycle regulation or in DNA replication. The DP2/E2F complex functions in the control of cell-cycle progression from G1 to S phase. The E2F1/DP complex appears to mediate both cell proliferation and apoptosis. Belongs to the E2F/DP family.
UniProt Protein Details:

Protein type:Transcription, coactivator/corepressor; DNA-binding

Chromosomal Location of Human Ortholog: 13q34

Cellular Component: nucleoplasm; nucleus

Molecular Function:protein binding; protein domain specific binding; transcription factor activity; transcription factor binding

Biological Process: cell proliferation; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; positive regulation of transcription from RNA polymerase II promoter; regulation of transcription from RNA polymerase II promoter

NCBI Summary:This gene encodes a member of a family of transcription factors that heterodimerize with E2F proteins to enhance their DNA-binding activity and promote transcription from E2F target genes. The encoded protein functions as part of this complex to control the transcriptional activity of numerous genes involved in cell cycle progression from G1 to S phase. Alternative splicing results in multiple transcript variants. Pseudogenes of this gene are found on chromosomes 1, 15, and X.[provided by RefSeq, Jan 2009]
UniProt Code:Q14186
NCBI GenInfo Identifier:3122926
NCBI Gene ID:7027
NCBI Accession:Q14186.1
UniProt Secondary Accession:Q14186,Q5JSB4, Q8IZL5, B4DLQ9,
UniProt Related Accession:Q14186
Molecular Weight:34,921 Da
NCBI Full Name:Transcription factor Dp-1
NCBI Synonym Full Names:transcription factor Dp-1
NCBI Official Symbol:TFDP1  
NCBI Official Synonym Symbols:DP1; DILC; Dp-1; DRTF1  
NCBI Protein Information:transcription factor Dp-1
UniProt Protein Name:Transcription factor Dp-1
UniProt Synonym Protein Names:DRTF1-polypeptide 1; DRTF1; E2F dimerization partner 1
Protein Family:Transcription factor
UniProt Gene Name:TFDP1  
UniProt Entry Name:TFDP1_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-DP-1 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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