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SIX6 Colorimetric Cell-Based ELISA

SKU:
CBCAB00963
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

SIX6 Colorimetric Cell-Based ELISA

The Six6 Colorimetric Cell-Based ELISA Kit is a cutting-edge product designed for the precise and efficient detection of six different cell-based ELISA targets in various biological samples. With its high sensitivity and specificity, this kit ensures accurate and reproducible results, making it a valuable tool for researchers in a wide range of fields.This kit allows researchers to analyze different cellular pathways and responses by measuring the levels of specific targets within cell lysates or culture supernatants. It is ideal for studying various biological processes, including cell signaling, proliferation, and apoptosis, providing valuable insights into cellular mechanisms and disease pathways.

Whether you are conducting basic research or drug development studies, the Six6 Colorimetric Cell-Based ELISA Kit offers a reliable and convenient solution for detecting multiple targets simultaneously, saving time and resources. Upgrade your research capabilities with this innovative kit and unlock new possibilities in cell-based assays.

Product Name:SIX6 Colorimetric Cell-Based ELISA
Product Code:CBCAB00963
ELISA Type:Cell-Based
Target:SIX6
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The SIX6 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SIX6 protein expression profile in cells. The kit can be used for measuring the relative amounts of SIX6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on SIX6.

Qualitative determination of SIX6 concentration is achieved by an indirect ELISA format. In essence, SIX6 is captured by SIX6-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: 4990, UniProt ID: O95475, OMIM: 606326, Unigene: Hs.194756
Gene Symbol:SIX6
Sub Type:None
UniProt Protein Function:SIX6: May be involved in eye development. Defects in SIX6 are the cause of microphthalmia isolated with cataract type 2 (MCOPCT2). Microphthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. Ocular abnormalities like opacities of the cornea and lens, scaring of the retina and choroid, cataractand other abnormalities like cataract may also be present. Belongs to the SIX/Sine oculis homeobox family.
UniProt Protein Details:

Protein type:DNA-binding

Chromosomal Location of Human Ortholog: 14q23.1

Biological Process: organ morphogenesis; visual perception

Disease: Microphthalmia, Isolated, With Cataract 2; Microphthalmia, Syndromic 3

NCBI Summary:The protein encoded by this gene is a homeobox protein that is similar to the Drosophila 'sine oculis' gene product. This gene is found in a cluster of related genes on chromosome 14 and is thought to be involved in eye development. Defects in this gene are a cause of isolated microphthalmia with cataract type 2 (MCOPCT2). [provided by RefSeq, Jul 2008]
UniProt Code:O95475
NCBI GenInfo Identifier:115502450
NCBI Gene ID:4990
NCBI Accession:O95475.2
UniProt Secondary Accession:O95475,Q6NT42, Q9P1X8,
UniProt Related Accession:O95475
Molecular Weight:27,687 Da
NCBI Full Name:Homeobox protein SIX6
NCBI Synonym Full Names:SIX homeobox 6
NCBI Official Symbol:SIX6  
NCBI Official Synonym Symbols:Six9; ODRMD; OPTX2; MCOPCT2  
NCBI Protein Information:homeobox protein SIX6
UniProt Protein Name:Homeobox protein SIX6
UniProt Synonym Protein Names:Homeodomain protein OPTX2; Optic homeobox 2; Sine oculis homeobox homolog 6
Protein Family:Homeobox protein
UniProt Gene Name:SIX6  
UniProt Entry Name:SIX6_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-SIX6 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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