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NCBP1 Colorimetric Cell-Based ELISA

SKU:
CBCAB01071
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

NCBP1 Colorimetric Cell-Based ELISA

The NCBP1 Colorimetric Cell-Based ELISA Kit is a powerful tool for studying the role of NCBP1 in cell biology. This kit allows for the quantitative measurement of NCBP1 levels in cell lysates, providing researchers with valuable insights into the function of this protein in cellular processes. NCBP1, also known as nuclear cap-binding protein subunit 1, plays a critical role in mRNA processing and transport, making it essential for gene expression regulation. Dysregulation of NCBP1 has been implicated in various diseases, including cancer and neurological disorders, highlighting its relevance as a potential therapeutic target.

The NCBP1 Colorimetric Cell-Based ELISA Kit offers high sensitivity and specificity, allowing for accurate and reproducible results. With its user-friendly protocol and efficient assay procedure, researchers can easily study the impact of NCBP1 on cellular pathways and disease mechanisms. This kit is a valuable resource for drug discovery, biomarker identification, and basic research in cell biology.

Product Name:NCBP1 Colorimetric Cell-Based ELISA
Product Code:CBCAB01071
ELISA Type:Cell-Based
Target:NCBP1
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The NCBP1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NCBP1 protein expression profile in cells. The kit can be used for measuring the relative amounts of NCBP1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NCBP1.

Qualitative determination of NCBP1 concentration is achieved by an indirect ELISA format. In essence, NCBP1 is captured by NCBP1-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: 4686, UniProt ID: Q09161, OMIM: 600469, Unigene: Hs.595669/Hs.686479
Gene Symbol:NCBP1
Sub Type:None
UniProt Protein Function:NCBP1: mediates U snRNA export from the nucleus. Binds to 5' capped mRNA. Found in aU snRNA export complex with RNUXA/PHAX, CBP20, CBP80, RAN, XPO1 and m7G-capped RNA. Interaction with RNUXA/PHAX. Heterodimer with NCBP2. Is part of the exon junction complex (EJC) containing NCBP1, NCBP2, RNPS1, RBM8A, SRRM1, NXF1, UPF3B, UPF2, THOC4 and/or REFBP2.
UniProt Protein Details:

Protein type:Translation; RNA processing; Spliceosome

Chromosomal Location of Human Ortholog: 9q34.1

Cellular Component: cytoplasm; cytosol; mitochondrion; mRNA cap complex; nucleoplasm; nucleus; ribonucleoprotein complex

Molecular Function:protein binding; RNA binding; RNA cap binding

Biological Process: gene expression; histone mRNA metabolic process; mRNA 3'-end processing; mRNA capping; mRNA catabolic process, nonsense-mediated decay; mRNA export from nucleus; nuclear mRNA cis splicing, via U2-type spliceosome; nuclear mRNA splicing, via spliceosome; positive regulation of cell growth; positive regulation of mRNA 3'-end processing; positive regulation of viral transcription; regulation of translational initiation; RNA elongation from RNA polymerase II promoter; RNA splicing; RNA-mediated gene silencing; spliceosomal snRNP biogenesis; termination of RNA polymerase II transcription; transcription from RNA polymerase II promoter; viral reproduction

NCBI Summary:The product of this gene is a component of the nuclear cap-binding protein complex (CBC), which binds to the monomethylated 5' cap of nascent pre-mRNA in the nucleoplasm. The encoded protein promotes high-affinity mRNA-cap binding and associates with the CTD of RNA polymerase II. The CBC promotes pre-mRNA splicing, 3'-end processing, RNA nuclear export, and nonsense-mediated mRNA decay. [provided by RefSeq, Jul 2008]
UniProt Code:Q09161
NCBI GenInfo Identifier:1705654
NCBI Gene ID:4686
NCBI Accession:Q09161.1
UniProt Secondary Accession:Q09161,Q59G76, Q5T1V0, Q5T7X2, B2R718,
UniProt Related Accession:Q09161
Molecular Weight:91,839 Da
NCBI Full Name:Nuclear cap-binding protein subunit 1
NCBI Synonym Full Names:nuclear cap binding protein subunit 1
NCBI Official Symbol:NCBP1  
NCBI Official Synonym Symbols:NCBP; Sto1; CBP80  
NCBI Protein Information:nuclear cap-binding protein subunit 1
UniProt Protein Name:Nuclear cap-binding protein subunit 1
UniProt Synonym Protein Names:80 kDa nuclear cap-binding protein; CBP80; NCBP 80 kDa subunit
Protein Family:CBP80/20-dependent translation initiation factor
UniProt Gene Name:NCBP1  
UniProt Entry Name:NCBP1_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-NCBP1 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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