Human GATA-2 Transcription Factor Activity Assay

$402.00
TFEH-GATA2-1

RayBio® Human GATA-2 Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical GATA-2 binding sequences to semi-quantitatively detect active GATA-2 in lysates or nuclear extracts. Dry ice shipment (additional fee). Only available in North America.

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Antigen Information

Accession Number:
  • P23769
Gene ID:
  • 2624
Gene Symbol:
  • GATA2
Protein Name & Synonyms:
Endothelial transcription factor GATA-2, GATA-binding protein 2
Target Species:
  • Human

Assay Format

Specificity:
The olionucleotide/antibody pair provided in this kit recognizes human FRA-2 in whole lysates and nuclear extracts.

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 to 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 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-2 from nuclear extracts of K562 cells or HeLa cells. A. Western-blot result of GATA-2 from cytoplasmic and nuclear fractions. B. Transcription factor activity assay of GATA-2 from nuclear fractions with the RayBio® GATA-2 TF-Activity Assay Kit.

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