GALK1 Colorimetric Cell-Based ELISA

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SKU:
CBCAB01026
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Metabolism
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
Frequently bought together:

Description

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GALK1 Colorimetric Cell-Based ELISA

The Human GALK1 Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate measurement of GALK1 levels in cell lysates and culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reproducible results for a variety of research applications.GALK1, also known as galactokinase 1, is an enzyme that plays a crucial role in galactose metabolism. Dysregulation of GALK1 has been implicated in various metabolic disorders, including galactosemia.

By measuring GALK1 levels, researchers can gain insights into galactose metabolism and its impact on metabolic diseases.This kit provides a user-friendly and cost-effective solution for studying GALK1 expression and function, making it an essential tool for researchers exploring galactose metabolism and its implications in human health and disease.

Product Name:GALK1 Colorimetric Cell-Based ELISA
Product Code:CBCAB01026
ELISA Type:Cell-Based
Target:GALK1
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The GALK1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GALK1 protein expression profile in cells. The kit can be used for measuring the relative amounts of GALK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GALK1.

Qualitative determination of GALK1 concentration is achieved by an indirect ELISA format. In essence, GALK1 is captured by GALK1-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: 2584, UniProt ID: P51570, OMIM: 230200/604313, Unigene: Hs.407966
Gene Symbol:GALK1
Sub Type:None
UniProt Protein Function:GALK1: Major enzyme for galactose metabolism. Defects in GALK1 are the cause of galactosemia II (GALCT2). Galactosemia II is an autosomal recessive deficiency characterized by congenital cataracts during infancy and presenile cataracts in the adult population. The cataracts are secondary to accumulation of galactitol in the lenses. Belongs to the GHMP kinase family. GalK subfamily.
UniProt Protein Details:

Protein type:Kinase, other; Carbohydrate Metabolism - galactose; EC 2.7.1.6; Carbohydrate Metabolism - amino sugar and nucleotide sugar

Chromosomal Location of Human Ortholog: 17q24

Cellular Component: cytoplasm; cytosol; membrane

Molecular Function:ATP binding; galactokinase activity; galactose binding

Biological Process: galactose catabolic process; galactose metabolic process

Disease: Galactokinase Deficiency

NCBI Summary:Galactokinase is a major enzyme for the metabolism of galactose and its deficiency causes congenital cataracts during infancy and presenile cataracts in the adult population. [provided by RefSeq, Jul 2008]
UniProt Code:P51570
NCBI GenInfo Identifier:1730187
NCBI Gene ID:2584
NCBI Accession:P51570.1
UniProt Secondary Accession:P51570,B2RC07, B4E1G6,
UniProt Related Accession:P51570
Molecular Weight:45,358 Da
NCBI Full Name:Galactokinase
NCBI Synonym Full Names:galactokinase 1
NCBI Official Symbol:GALK1  
NCBI Official Synonym Symbols:GK1; GALK; HEL-S-19  
NCBI Protein Information:galactokinase
UniProt Protein Name:Galactokinase
UniProt Synonym Protein Names:Galactose kinase
UniProt Gene Name:GALK1  
UniProt Entry Name:GALK1_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-GALK1 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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