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Kir6.2 (Phospho-Thr224) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01461
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Signal Transduction
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

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Kir6.2 (Phospho-Thr224)Colorimetric Cell-Based ELISA Kit

The KIR6.2 Phospho-Thr224 Colorimetric Cell-based ELISA Kit is a cutting-edge tool for researchers looking to accurately detect and quantify levels of phosphorylated KIR6.2 protein in cell lysates and tissue samples. This kit offers high sensitivity and specificity, ensuring precise and reproducible results for a variety of experimental needs.KIR6.2 is a key component of ATP-sensitive potassium channels, playing a crucial role in regulating cellular excitability and insulin secretion. Phosphorylation of KIR6.2 at Thr224 is known to modulate channel activity, making it an important target for studying the physiological and pathological roles of these channels in conditions such as diabetes and cardiac disorders.

With its easy-to-use colorimetric assay format and comprehensive instructions, the KIR6.2 Phospho-Thr224 Colorimetric Cell-based ELISA Kit is an essential tool for researchers working in the fields of cellular signaling, ion channel biology, and disease mechanisms. Get reliable and accurate data with this innovative kit from Assay Genie.

Product Name:Kir6.2 (Phospho-Thr224) Colorimetric Cell-Based ELISA
Product Code:CBCAB01461
ELISA Type:Cell-Based
Target:Kir6.2 (Phospho-Thr224)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Kir6.2 (Phospho-Thr224) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Kir6.2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Kir6.2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Kir6.2 phosphorylation.

Qualitative determination of Kir6.2 (Phospho-Thr224) concentration is achieved by an indirect ELISA format. In essence, Kir6.2 (Phospho-Thr224) is captured by Kir6.2 (Phospho-Thr224)-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: 3767, UniProt ID: Q14654, OMIM: 600937/601820/606176/610582, Unigene: Hs.248141
Gene Symbol:KCNJ11
Sub Type:Phospho
UniProt Protein Function:Kir6.2: This receptor is controlled by G proteins. Inward rectifier potassium channels are characterized by a greater tendency to allow potassium to flow into the cell rather than out of it. Their voltage dependence is regulated by the concentration of extracellular potassium; as external potassium is raised, the voltage range of the channel opening shifts to more positive voltages. The inward rectification is mainly due to the blockage of outward current by internal magnesium. Can be blocked by extracellular barium. Subunit of ATP-sensitive potassium channels (KATP). Can form cardiac and smooth muscle-type KATP channels with ABCC9. KCNJ11 forms the channel pore while ABCC9 is required for activation and regulation. Defects in KCNJ11 are the cause of familial hyperinsulinemic hypoglycemia type 2 (HHF2); also known as persistent hyperinsulinemic hypoglycemia of infancy (PPHI) or congenital hyperinsulinism. HHF is the most common cause of persistent hypoglycemia in infancy and is due to defective negative feedback regulation of insulin secretion by low glucose levels. It causes nesidioblastosis, a diffuse abnormality of the pancreas in which there is extensive, often disorganized formation of new islets. Unless early and aggressive intervention is undertaken, brain damage from recurrent episodes of hypoglycemia may occur. Defects in KCNJ11 are a cause of diabetes mellitus permanent neonatal (PNDM). PNDM is a rare form of diabetes distinct from childhood-onset autoimmune diabetes mellitus type 1. It is characterized by insulin-requiring hyperglycemia that is diagnosed within the first months of life. Permanent neonatal diabetes requires lifelong therapy. Defects in KCNJ11 are the cause of transient neonatal diabetes mellitus type 3 (TNDM3). Neonatal diabetes mellitus, defined as insulin-requiring hyperglycemia within the first month of life, is a rare entity. In about half of the neonates, diabetes is transient and resolves at a median age of 3 months, whereas the rest have a permanent form of diabetes. In a significant number of patients with transient neonatal diabetes mellitus, diabetes type 2 appears later in life. The onset and severity of TNDM3 is variable with childhood-onset diabetes, gestational diabetes or adult-onset diabetes described. Defects in KCNJ11 may contribute to non-insulin- dependent diabetes mellitus (NIDDM), also known as diabetes mellitus type 2. Belongs to the inward rectifier-type potassium channel (TC 1.A.2.1) family. KCNJ11 subfamily.
UniProt Protein Details:

Protein type:Membrane protein, multi-pass; Membrane protein, integral; Channel, potassium

Chromosomal Location of Human Ortholog: 11p15.1

Cellular Component: ATP-sensitive potassium channel complex; integral to plasma membrane; plasma membrane; T-tubule; voltage-gated potassium channel complex

Molecular Function:ankyrin binding; ATP binding; ATP-activated inward rectifier potassium channel activity; potassium ion binding; voltage-gated potassium channel activity

Biological Process: glucose metabolic process; negative regulation of insulin secretion; neurological system process; potassium ion import; regulation of insulin secretion; regulation of membrane potential; response to ATP; response to drug; transmembrane transport

Disease: Diabetes Mellitus, Noninsulin-dependent; Diabetes Mellitus, Permanent Neonatal; Diabetes Mellitus, Transient Neonatal, 3; Hyperinsulinemic Hypoglycemia, Familial, 2; Maturity-onset Diabetes Of The Young, Type 13

NCBI Summary:Potassium channels are present in most mammalian cells, where they participate in a wide range of physiologic responses. The protein encoded by this gene is an integral membrane protein and inward-rectifier type potassium channel. The encoded protein, which has a greater tendency to allow potassium to flow into a cell rather than out of a cell, is controlled by G-proteins and is found associated with the sulfonylurea receptor SUR. Mutations in this gene are a cause of familial persistent hyperinsulinemic hypoglycemia of infancy (PHHI), an autosomal recessive disorder characterized by unregulated insulin secretion. Defects in this gene may also contribute to autosomal dominant non-insulin-dependent diabetes mellitus type II (NIDDM), transient neonatal diabetes mellitus type 3 (TNDM3), and permanent neonatal diabetes mellitus (PNDM). Multiple alternatively spliced transcript variants that encode different protein isoforms have been described for this gene. [provided by RefSeq, Oct 2009]
UniProt Code:Q14654
NCBI GenInfo Identifier:76803775
NCBI Gene ID:3767
NCBI Accession:Q14654.2
UniProt Secondary Accession:Q14654,Q2M1H7, Q58EX3, Q8IW96, B4DWI4, E9PNK0,
UniProt Related Accession:Q14654
Molecular Weight:33,263 Da
NCBI Full Name:ATP-sensitive inward rectifier potassium channel 11
NCBI Synonym Full Names:potassium voltage-gated channel subfamily J member 11
NCBI Official Symbol:KCNJ11  
NCBI Official Synonym Symbols:BIR; HHF2; PHHI; IKATP; TNDM3; KIR6.2; MODY13  
NCBI Protein Information:ATP-sensitive inward rectifier potassium channel 11
UniProt Protein Name:ATP-sensitive inward rectifier potassium channel 11
UniProt Synonym Protein Names:IKATP; Inward rectifier K(+) channel Kir6.2; Potassium channel, inwardly rectifying subfamily J member 11
Protein Family:ATP-sensitive inward rectifier potassium channel
UniProt Gene Name:KCNJ11  
UniProt Entry Name:KCJ11_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-Kir6.2 (Phospho-Thr224) Antibody, Anti-Kir6.2 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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