Human GATA-1 Transcription Factor Activity Assay

RayBio® Human GATA-1 Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical GATA1 binding sequences to semi-quantitatively detect active GATA1 in lysates or nuclear extracts. Dry ice shipment (additional fee).

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TFEH-GATA1-1
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Antigen Information

Accession Number:
  • P15976
Gene ID:
  • 2623
Gene Symbols:
  • ERYF1
  • GATA1
  • GF1
Protein Name & Synonyms:
Erythroid transcription factor Eryf1 GATA-binding factor 1 (GATA-1, GF-1) NF-E1 DNA-binding protein
Target Species:
  • Human

Assay Format

Specificity:
The olionucleotide/antibody pair provided in this kit recognizes human GATA-1 in whole lysates and nuclear extracts.
Number of Targets Detected:
1
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Nuclear Extracts
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

GATA transcription factors have a characteristic feature that they all bind to the DNA consensus sequence (A/T)GATA(A/G) of target gene promoters by two adjacent zinc fingers (Cys-X2-Cys-X17-Cys-X2-Cys). These transcription factors regulate differentiation, growth and survival of a wide range of cell types. The GATA family consists of six transcription factors, GATA1 to GATA6, between which the DNA-binding regions, two zinc finger domains are highly conserved. The C-terminal €˜€˜C-finger€™€™ and its adjacent basic region are necessary and sufficient for GATA to bind its cognate sequence, The N-terminal €˜€˜N-finger€™€™ can also bind DNA independently but has a preference for GATC core motifs. Both fingers participate in binding to the palindromic GATA motif ATCWGATA (W = A/T), resulting in markedly increased affinity.

Product Features

  • Specific transcription factor-DNA binding assay
  • Perfect alternative to EMSA
  • Easy to perform in an ELISA format
  • Non-radioactive assay
  • High throughput (96-well plate format)
  • Assay can be completed within 5 hours

Application Notes

Kit Components
  • 96-well Strip Microplate pre-coated with DNA probes
  • DNA Binding Buffer
  • Positive Control Sample
  • Specific Competitor DNA probe
  • Non-specific Competitor DNA probe
  • Assay Reagent
  • DTT
  • Wash Buffer
  • Primary Antibody
  • HRP-conjugated Secondary Antibody
  • Antibody Diluent Buffer
  • TMB One-Step Substrate Reagent
  • Stop Solution
Other Materials Required
  • Distilled or deionized water
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare sample dilutions
  • Absorbent paper
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared HRP-secondary antibody to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Figure 1
Transcription factor activity assay of GATA-1 from nuclear extracts of K562 cells or HeLa cells. In K562 cells, activated GATA-1 is translocated into the nucleus where it binds with its corresponding DNA. A. Western-blot result of GATA-1 from cytoplasmic and nuclear fractions. B. Transcription factor activity assay of GATA-1 from nuclear fractions with the RayBio® GATA-1 Transcription Factor-Activity Assay Kit.

Figure 2
Transcription factor activity assay of GATA-1 from nuclear extracts of K562 cells or HeLa cells with the specific competitor or non-specific competitor. The result shows specific binding of GATA-1 to the GATA conserved binding site detected by using the RayBio® GATA-1 Transcription Factor-Activity Assay Kit.

Storage/Stability

Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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