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HOXA1 Colorimetric Cell-Based ELISA

SKU:
CBCAB01018
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
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Description

system_update_altDatasheetMSDS

HOXA1 Colorimetric Cell-Based ELISA

The HOXA1 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the accurate measurement of HOXA1 protein levels in cell lysates and tissue samples. This kit offers high sensitivity and specificity, allowing for precise and reproducible results in a variety of research applications.HOXA1 is a key transcription factor known to regulate embryonic development and cell differentiation. Dysregulation of HOXA1 has been linked to various diseases including cancer, developmental disorders, and neurological conditions.

By measuring HOXA1 levels, researchers can gain valuable insights into the molecular mechanisms underlying these diseases and potentially identify new therapeutic targets.With its user-friendly protocol and reliable performance, the HOXA1 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers studying HOXA1 function and its implications in human health and disease.

Product Name:HOXA1 Colorimetric Cell-Based ELISA
Product Code:CBCAB01018
ELISA Type:Cell-Based
Target:HOXA1
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The HOXA1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HOXA1 protein expression profile in cells. The kit can be used for measuring the relative amounts of HOXA1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on HOXA1.

Qualitative determination of HOXA1 concentration is achieved by an indirect ELISA format. In essence, HOXA1 is captured by HOXA1-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: 3198, UniProt ID: P49639, OMIM: 142955/601536, Unigene: Hs.67397
Gene Symbol:HOXA1
Sub Type:None
UniProt Protein Function:HOXA1: Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. Acts on the anterior body structures. Seems to act in the maintenance and/or generation of hindbrain segments. Defects in HOXA1 are the cause of Athabaskan brainstem dysgenesis syndrome (ABDS); also known as Narvajo brainstem syndrome. This syndrome is characterized by horizontal gaze palsy, sensorineural deafness, central hypoventilation, and developmental delay. Some patients had swallowing dysfunction, vocal cord paralysis, facial paresis, seizures, and cardiac outflow tract anomalies. Defects in HOXA1 are the cause of Bosley-Salih-Alorainy syndrome (BSAS). Affected individuals show horizontal gaze abnormalities, deafness, facial weakness, vascular malformations of the internal carotid arteries and cardiac outflow trac. Some patients manifest mental retardation and autism spectrum disorder. In contrast to individuals with ABSD, central hypoventilation is not observed in individuals with BSAS. Belongs to the Antp homeobox family. Labial subfamily. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Oncoprotein; DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 7p15.3

Molecular Function:protein binding

Biological Process: abducens nerve formation; anatomical structure morphogenesis; artery morphogenesis; cognition; embryonic neurocranium morphogenesis; inner ear development; multicellular organismal development; neuromuscular process; optokinetic behavior; outer ear morphogenesis; regulation of behavior; sensory perception of sound

Disease: Athabaskan Brainstem Dysgenesis Syndrome

NCBI Summary:In vertebrates, the genes encoding the class of transcription factors called homeobox genes are found in clusters named A, B, C, and D on four separate chromosomes. Expression of these proteins is spatially and temporally regulated during embryonic development. This gene is part of the A cluster on chromosome 7 and encodes a DNA-binding transcription factor which may regulate gene expression, morphogenesis, and differentiation. The encoded protein may be involved in the placement of hindbrain segments in the proper location along the anterior-posterior axis during development. Two transcript variants encoding two different isoforms have been found for this gene, with only one of the isoforms containing the homeodomain region. [provided by RefSeq, Jul 2008]
UniProt Code:P49639
NCBI GenInfo Identifier:6166216
NCBI Gene ID:3198
NCBI Accession:P49639.2
UniProt Secondary Accession:P49639,O43363, A4D184, B2R8U7,
UniProt Related Accession:P49639
Molecular Weight:24,489 Da
NCBI Full Name:Homeobox protein Hox-A1
NCBI Synonym Full Names:homeobox A1
NCBI Official Symbol:HOXA1  
NCBI Official Synonym Symbols:BSAS; HOX1; HOX1F  
NCBI Protein Information:homeobox protein Hox-A1
UniProt Protein Name:Homeobox protein Hox-A1
UniProt Synonym Protein Names:Homeobox protein Hox-1F
Protein Family:Homeobox protein
UniProt Gene Name:HOXA1  
UniProt Entry Name:HXA1_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-HOXA1 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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