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GATA1 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00671
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheet

GATA1 Colorimetric Cell-Based ELISA Kit

The GATA1 Colorimetric Cell-Based ELISA Kit is a powerful tool designed for the sensitive and accurate detection of GATA1 levels in cell lysates and tissue homogenates. This kit offers high specificity and reproducibility, allowing for reliable results in a variety of research applications.GATA1 is a critical transcription factor that plays a key role in the regulation of gene expression, particularly in the development and differentiation of hematopoietic cells.

Dysregulation of GATA1 has been linked to various hematological disorders, including leukemia and anemia, highlighting its importance as a biomarker for studying these conditions.With its user-friendly protocol and excellent performance, the GATA1 Colorimetric Cell-Based ELISA Kit is a valuable tool for researchers looking to explore the role of GATA1 in health and disease.

Product Name:GATA1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00671
ELISA Type:Cell-Based
Target:GATA1
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The GATA1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GATA1 protein expression profile in cells. The kit can be used for measuring the relative amounts of GATA1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GATA1.

Qualitative determination of GATA1 concentration is achieved by an indirect ELISA format. In essence, GATA1 is captured by GATA1-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: 2623, UniProt ID: P15976, OMIM: 300367/305371/314050, Unigene: Hs.765
Gene Symbol:GATA1
Sub Type:None
UniProt Protein Function:Transcriptional activator or repressor which probably serves as a general switch factor for erythroid development. It binds to DNA sites with the consensus sequence 5'-[AT]GATA[AG]-3' within regulatory regions of globin genes and of other genes expressed in erythroid cells. Activates the transcription of genes involved in erythroid differentiation of K562 erythroleukemia cells, including HBB, HBG1/2, ALAS2 and HMBS (PubMed:24245781).
NCBI Summary:This gene encodes a protein which belongs to the GATA family of transcription factors. The protein plays an important role in erythroid development by regulating the switch of fetal hemoglobin to adult hemoglobin. Mutations in this gene have been associated with X-linked dyserythropoietic anemia and thrombocytopenia. [provided by RefSeq, Jul 2008]
UniProt Code:P15976
NCBI GenInfo Identifier:120956
NCBI Gene ID:2623
NCBI Accession:P15976.1
UniProt Secondary Accession:P15976,Q96GB8,
UniProt Related Accession:P15976
Molecular Weight:34,232 Da
NCBI Full Name:Erythroid transcription factor
NCBI Synonym Full Names:GATA binding protein 1
NCBI Official Symbol:GATA1  
NCBI Official Synonym Symbols:GF1; GF-1; NFE1; XLTT; ERYF1; NF-E1; XLANP; XLTDA; GATA-1  
NCBI Protein Information:erythroid transcription factor
UniProt Protein Name:Erythroid transcription factor
UniProt Synonym Protein Names:Eryf1; GATA-binding factor 1; GATA-1; GF-1; NF-E1 DNA-binding protein
Protein Family:GATA transcription factor
UniProt Gene Name:GATA1  
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-GATA1 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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