K6PP Colorimetric Cell-Based ELISA

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SKU:
CBCAB01057
€499

Description

K6PP Cell-Based ELISA Kit

The K6PP Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor K6PP protein expression profile in cells. The kit can be used for measuring the relative amounts of K6PP in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on K6PP .

How does our K6PP Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of K6PP concentration is achieved by an indirect ELISA format. In essence, K6PP is captured by K6PP -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° 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.

K6PP Colorimetric Cell-Based ELISA - Information

Product Name:K6PP Colorimetric Cell-Based ELISA
Product Code:/SKUCBCAB01057
Description:The K6PP Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect K6PP protein expression profile in cells. The kit can be used for measuring the relative amounts of K6PP in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on K6PP.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based
Database Links:Gene ID: 5214, UniProt ID: Q01813, OMIM #: 171840, Unigene #: Hs.26010
Format:96-Well Microplate
NCBI Gene Symbol:K6PP
Sub Type:None
Target Name:K6PP

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

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 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
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

Additional equipment and materials required

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

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

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-K6PP 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.)

K6PP - Protein Information

UniProt Protein Function:PFKP: phosphofructokinase, platelet type. An ubiquitous metabolic enzyme involved in the synthesis and degradation of fructose 2,6-bisphosphate. Key control step of glycolysis. An allosteric enzyme activated by ADP, AMP, or fructose bisphosphate and inhibited by ATP or citrate. Activity: ATP D-fructose 6-phosphate = ADP D-fructose 1,6-bisphosphate. The holoenzyme consists of 4 subunits. The liver and muscle enzymes are homo-tetramers of four liver or muscle isoforms, respectively. The red blood cell enzyme consists hetero-tetramers of the muscle and liver isoforms. A subunit composition with a higher proportion of platelet type subunits is found in platelets, brain and fibroblasts.
UniProt Protein Details:

Protein type:Carbohydrate Metabolism - glycolysis and gluconeogenesis; Carbohydrate Metabolism - pentose phosphate pathway; EC 2.7.1.11; Carbohydrate Metabolism - galactose; Kinase, other; Carbohydrate Metabolism - fructose and mannose

Chromosomal Location of Human Ortholog: 10p15.3-p15.2

Cellular Component: cytosol; membrane; nucleus

Molecular Function:6-phosphofructokinase activity; protein complex binding

NCBI Summary:The PFKP gene encodes the platelet isoform of phosphofructokinase (PFK) (ATP:D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11). PFK catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. The PFKP gene, which maps to chromosome 10p, is also expressed in fibroblasts. See also the muscle (PFKM; MIM 610681) and liver (PFKL; MIM 171860) isoforms of phosphofructokinase, which map to chromosomes 12q13 and 21q22, respectively. Vora (1981) [PubMed 6451249] determined that full tetrameric phophofructokinase enzyme expressed in platelets can be composed of subunits P4, P3L, and P2L2.[supplied by OMIM, Mar 2008]
UniProt Code:Q01813
NCBI GenInfo Identifier:1346355
NCBI Gene ID:5214
NCBI Accession:Q01813.2
UniProt Secondary Accession:Q01813,Q5VSR7, Q5VSR8, B3KS15,
UniProt Related Accession:Q01813
Molecular Weight:85596 Da
NCBI Full Name:ATP-dependent 6-phosphofructokinase, platelet type
NCBI Synonym Full Names:phosphofructokinase, platelet
NCBI Official Symbol:PFKP  
NCBI Official Synonym Symbols:PFKF; PFK-C; PFK-P; ATP-PFK  
NCBI Protein Information:ATP-dependent 6-phosphofructokinase, platelet type
UniProt Protein Name:ATP-dependent 6-phosphofructokinase, platelet type
UniProt Synonym Protein Names:6-phosphofructokinase type C; Phosphofructo-1-kinase isozyme C; PFK-C; Phosphohexokinase
Protein Family:ATP-dependent 6-phosphofructokinase
UniProt Gene Name:PFKP  
UniProt Entry Name:PFKAP_HUMAN
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