Human Sp1 Transcription Factor Activity Assay

RayBio® Human Sp1 Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical Sp1 binding sequences to semi-quantitatively detect active Sp1 in lysates or nuclear extracts. Dry ice shipment (additional fee).

$1,608.00  
TFEH-SP1-5
+

Antigen Information

Accession Number:
  • P08047
Gene ID:
  • 6667
Gene Symbols:
  • SP1
  • TSFP1
Protein Name & Synonyms:
Transcription factor Sp1
Target Species:
  • Human

Assay Format

Pathway:
  • JAK/STAT Signaling
  • p53 Signaling
Specificity:
The olionucleotide/antibody pair provided in this kit recognizes human Sp1 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

Sp1 is a 785-amino-acid, 100- to 110-kDa nuclear transcription factor belonging to a specific protein family which has 8 (Sp1-Sp8) members. It is ubiquitously expressed and regulates the expression of thousands of genes implicated in the control of a diverse array of cellular processes, such as cell growth, differentiation, apoptosis, angiogenesis, and immune response, via multiple mechanisms in response to physiologic and pathological stimuli. Sp1 possesses C2H2-type zinc finger domain through which it binds GC-rich motifs (such as 5'-G/T-GGGCGG-G/A-G/A-C/T-3' or 5'-G/T-G/A-GGCG-G/T-G/A-G/A-C/T-3') of a DNA sequence in promoters of target genes with high affinity. It can also regulate the expression of genes containing and not containging TATA and TATA genes via protein-protein interactions or interplay with other transcription factors, such as Ets-1, c-myc, c-Jun, Stat1, and Egr-1, and/or components of the basal transcriptional machinery. Sp1 has been linked to chromatin remodeling through interactions with chromatin-modifying factors such as p300 and histone deacetylases (HDACs). It is expected that posttranslational modifications including phosphorylation, acetylation, sumoylation, ubiquitylation, and glycosylation can influence the transcriptional activity and stability of Sp1.

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 assay of Sp1 from nuclear extracts of HeLa cells or HeLa cells treated with PMA (50 ng/ml) for 3 hr with the RayBio® Activity Assay Kit.

Transcription factor assay of Sp1 from nuclear extracts of HeLa cells or HeLa cells treated with PMA (50 ng/ml) for 3 hr with the RayBio Activity Assay Kit

Figure 2
Transcription factor assay of Sp1 from nuclear extracts of HeLa cells or HeLa cells treated with PMA (50 ng/ml) for 3 hr with the specific competitor or non-specific competitor. The result shows specific binding of Sp1 to the conserve binding site detected by using the RayBio® Sp1 TF Activity Assay Kit .

Transcription factor assay of Sp1 from nuclear extracts of HeLa cells or HeLa cells treated with PMA (50 ng/ml) for 3 hr with the specific competitor or non-specific competitor. The result shows specific binding of Sp1 to the conserve binding site detected by using the RayBio Sp1 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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