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). 6 - 8 week lead time.
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
<lIκBenchtop rocker or shaker
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 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.
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 .
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.
