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NIPA (Phospho-Ser354) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01472
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Cycle
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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NIPA (Phospho-Ser354)Colorimetric Cell-Based ELISA Kit

The NIPA Phospho-Ser354 Colorimetric Cell-Based ELISA Kit is specifically designed for the sensitive and accurate detection of phosphorylated NIPA (Non-Imprinted in Prader-Willi/Angelman Syndrome) protein at serine 354 in cell lysates. This kit offers high specificity and reproducibility, ensuring precise results for your research needs.Phosphorylation of the NIPA protein at serine 354 plays a crucial role in various cellular processes, including cell growth, proliferation, and differentiation. This kit allows researchers to study the phosphorylation status of NIPA protein in different cell types and conditions, providing valuable insights into its role in cellular signaling pathways.

With its user-friendly protocol and reliable performance, the NIPA Phospho-Ser354 Colorimetric Cell-Based ELISA Kit is an essential tool for studying the regulatory mechanisms of NIPA protein phosphorylation and its impact on cellular function. Order yours today and unlock the potential of your research projects.

Product Name:NIPA (Phospho-Ser354) Colorimetric Cell-Based ELISA
Product Code:CBCAB01472
ELISA Type:Cell-Based
Target:NIPA (Phospho-Ser354)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The NIPA (Phospho-Ser354) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NIPA protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated NIPA in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on NIPA phosphorylation.

Qualitative determination of NIPA (Phospho-Ser354) concentration is achieved by an indirect ELISA format. In essence, NIPA (Phospho-Ser354) is captured by NIPA (Phospho-Ser354)-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: 51530, UniProt ID: Q86WB0, OMIM: None, Unigene: Hs.194157
Gene Symbol:ZC3HC1
Sub Type:Phospho
UniProt Protein Function:NIPA: a nuclear protein that interacts with anaplastic lymphoma kinase (ALK). An essential component of an SCF-type E3 ligase complex, SCF(NIPA), a complex that controls mitotic entry by mediating ubiquitination and subsequent degradation of cyclin B1. Its cell-cycle-dependent phosphorylation regulates the assembly of the SCF(NIPA) complex, restricting cyclin B1 ubiquitination activity to interphase. Its inactivation results in nuclear accumulation of cyclin B1 in interphase and premature mitotic entry. May have an antiapoptotic role in NPM-ALK-mediated signaling events. Interacts with the NPM-ALK fusion protein in a tyrosine phosphorylation-dependent manner. Three splice-variant isoforms have been described.
UniProt Protein Details:

Protein type:Cell cycle regulation; Ubiquitin conjugating system

Chromosomal Location of Human Ortholog: 7q32.2

Cellular Component: nuclear membrane; nucleus

Molecular Function:protein binding; protein kinase binding; zinc ion binding

Biological Process: cell division; mitosis; protein ubiquitination

NCBI Summary:This gene encodes an F-box-containing protein that is a component of an SCF-type E3 ubiquitin ligase complex that regulates the onset of cell division. The G2/M transition in the cell cycle requires the interaction of the proteins cyclin B1 and cyclin-dependent kinase 1. The activated ubiquitin ligase complex targets the protein cyclin B1 for degradation, preventing this transition to mitosis. [provided by RefSeq, Aug 2013]
UniProt Code:Q86WB0
NCBI GenInfo Identifier:73921220
NCBI Gene ID:51530
NCBI Accession:Q86WB0.1
UniProt Secondary Accession:Q86WB0,Q75MF3, Q75MF4, Q8N330, Q96F75, Q9HA34, Q9NVX4 Q9P0R0, A6NH66,
UniProt Related Accession:Q86WB0
Molecular Weight:47,771 Da
NCBI Full Name:Nuclear-interacting partner of ALK
NCBI Synonym Full Names:zinc finger C3HC-type containing 1
NCBI Official Symbol:ZC3HC1  
NCBI Official Synonym Symbols:NIPA; HSPC216  
NCBI Protein Information:nuclear-interacting partner of ALK
UniProt Protein Name:Nuclear-interacting partner of ALK
UniProt Synonym Protein Names:Nuclear-interacting partner of anaplastic lymphoma kinase; hNIPA; Zinc finger C3HC-type protein 1
Protein Family:NIPA-like protein
UniProt Gene Name:ZC3HC1  
UniProt Entry Name:NIPA_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-NIPA (Phospho-Ser354) Antibody, Anti-NIPA 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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