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IPMK Colorimetric Cell-Based ELISA

SKU:
CBCAB01158
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Metabolism
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

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IPMK Colorimetric Cell-Based ELISA

The IPMK Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers interested in studying the IPMK protein in cell culture samples. With high sensitivity and specificity, this kit provides accurate and reliable results, making it ideal for a variety of research applications.IPMK, or inositol polyphosphate multikinase, is a key enzyme involved in cellular signaling pathways, particularly in the regulation of cell growth, proliferation, and metabolism. Dysregulation of IPMK has been linked to various diseases, including cancer, diabetes, and neurological disorders, highlighting its importance as a potential therapeutic target.

By detecting and quantifying IPMK levels in cell culture supernatants, this ELISA kit enables researchers to explore the role of this protein in disease development and progression, paving the way for the development of novel treatments and interventions. Trust in the IPMK Colorimetric Cell-Based ELISA Kit for accurate and precise measurement of IPMK levels in your research studies.

Product Name:IPMK Colorimetric Cell-Based ELISA
Product Code:CBCAB01158
ELISA Type:Cell-Based
Target:IPMK
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of IPMK concentration is achieved by an indirect ELISA format. In essence, IPMK is captured by IPMK-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: 253430, UniProt ID: Q8NFU5, OMIM: 609851, Unigene: Hs.499690
Gene Symbol:IPMK
Sub Type:None
UniProt Protein Function:IPMK: Inositol phosphate kinase with a broad substrate specificity. Has a preference for inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4). Belongs to the inositol phosphokinase (IPK) family.
UniProt Protein Details:

Protein type:Carbohydrate Metabolism - inositol phosphate; EC 2.7.1.151; Kinase, other; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 10q21.1

Cellular Component: nucleolus; nucleoplasm; nucleus

Molecular Function:ATP binding; inositol tetrakisphosphate 3-kinase activity; inositol trisphosphate 3-kinase activity; inositol trisphosphate 6-kinase activity

Biological Process: inositol phosphate biosynthetic process; inositol phosphate metabolic process; neural tube formation; phosphorylation

NCBI Summary:This gene encodes a member of the inositol phosphokinase family. The encoded protein has 3-kinase, 5-kinase and 6-kinase activities on phosphorylated inositol substrates. The encoded protein plays an important role in the biosynthesis of inositol 1,3,4,5,6-pentakisphosphate, and has a preferred 5-kinase activity. This gene may play a role in nuclear mRNA export. Pseudogenes of this gene are located on the long arm of chromosome 13 and the short arm of chromosome 19. [provided by RefSeq, Dec 2010]
UniProt Code:Q8NFU5
NCBI GenInfo Identifier:50401072
NCBI Gene ID:253430
NCBI Accession:Q8NFU5.1
UniProt Related Accession:Q8NFU5
Molecular Weight:47,222 Da
NCBI Full Name:Inositol polyphosphate multikinase
NCBI Synonym Full Names:inositol polyphosphate multikinase
NCBI Official Symbol:IPMK  
NCBI Protein Information:inositol polyphosphate multikinase
UniProt Protein Name:Inositol polyphosphate multikinase
UniProt Synonym Protein Names:Inositol 1,3,4,6-tetrakisphosphate 5-kinase
Protein Family:Inositol polyphosphate multikinase
UniProt Gene Name:IPMK  
UniProt Entry Name:IPMK_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-IPMK 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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