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HGS Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00691
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Biology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

HGS Colorimetric Cell-Based ELISA Kit

The HGS Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers looking to accurately detect levels of specific proteins in human cell cultures. This kit boasts high sensitivity and specificity, providing reliable and reproducible results for a variety of research applications.Whether studying angiogenesis, cancer, cardiovascular diseases, or neurodegenerative disorders, this kit is designed to help researchers uncover important insights into the role of angiogenin and other critical proteins in these conditions.

With its user-friendly format and precise measurement capabilities, the HGS Colorimetric Cell-Based ELISA Kit is a valuable asset for any laboratory looking to advance their research in the field of cell biology.

Product Name:HGS Colorimetric Cell-Based ELISA
Product Code:CBCAB00691
ELISA Type:Cell-Based
Target:HGS
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of HGS concentration is achieved by an indirect ELISA format. In essence, HGS is captured by HGS-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: 9146, UniProt ID: O14964, OMIM: 604375, Unigene: Hs.514590
Gene Symbol:HGS
Sub Type:None
UniProt Protein Function:Hrs: a zinc finger protein which is rapidly tyrosine phosphorylated in cells stimulated with various growth factors. Hrs mRNA is expressed in all the human adult and fetal tissues tested.
UniProt Protein Details:

Protein type:Adaptor/scaffold; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 17q25

Cellular Component: cytoplasm; cytosol; early endosome; early endosome membrane; endosome; intracellular membrane-bound organelle

Molecular Function:metal ion binding; protein binding; protein domain specific binding

Biological Process: autophagy; endosome transport; epidermal growth factor receptor signaling pathway; membrane invagination; negative regulation of cell proliferation; negative regulation of epidermal growth factor receptor signaling pathway; negative regulation of JAK-STAT cascade; post-Golgi vesicle-mediated transport; protein targeting to lysosome; regulation of MAP kinase activity; regulation of protein catabolic process; signal transduction

NCBI Summary:The protein encoded by this gene regulates endosomal sorting and plays a critical role in the recycling and degradation of membrane receptors. The encoded protein sorts monoubiquitinated membrane proteins into the multivesicular body, targeting these proteins for lysosome-dependent degradation. [provided by RefSeq, Dec 2010]
UniProt Code:O14964
NCBI GenInfo Identifier:71152119
NCBI Gene ID:9146
NCBI Accession:O14964.1
UniProt Secondary Accession:O14964,Q9NR36,
UniProt Related Accession:O14964
Molecular Weight:76,376 Da
NCBI Full Name:Hepatocyte growth factor-regulated tyrosine kinase substrate
NCBI Synonym Full Names:hepatocyte growth factor-regulated tyrosine kinase substrate
NCBI Official Symbol:HGS  
NCBI Official Synonym Symbols:HRS  
NCBI Protein Information:hepatocyte growth factor-regulated tyrosine kinase substrate
UniProt Protein Name:Hepatocyte growth factor-regulated tyrosine kinase substrate
UniProt Synonym Protein Names:Hrs
Protein Family:Hepatocyte growth factor-regulated tyrosine kinase substrate
UniProt Gene Name:HGS  
UniProt Entry Name:HGS_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-HGS 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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