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4000-520-616 / 18915418616
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0512-67156496
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info@ebiomall.com
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商品描述
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
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Typical Data
Figure 1Transcription 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 2Transcription 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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