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SP1 (Phospho-Thr453) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01320
Product type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Biology
€599
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Description

system_update_altDatasheet
Product Name:SP1 (Phospho-Thr453) Colorimetric Cell-Based ELISA
Product Code:CBCAB01320
ELISA Type:Cell-Based
Target:SP1 (Phospho-Thr453)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The SP1 (Phospho-Thr453) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SP1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SP1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SP1 phosphorylation.

Qualitative determination of SP1 (Phospho-Thr453) concentration is achieved by an indirect ELISA format. In essence, SP1 (Phospho-Thr453) is captured by SP1 (Phospho-Thr453)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 6667, UniProt ID: P08047, OMIM: 189906, Unigene: Hs.620754/Hs.649191
Gene Symbol:SP1
Sub Type:Phospho
UniProt Protein Function:SP1: a transcription factor of the Sp1 C2H2-type zinc-finger protein family. Phosphorylated and activated by MAPK. Dephosphorylation by PTEN inhibits DNA binding. Binds to p38 in the nucleus. Interacts with Huntingtin and TAFII130. Transcriptional activity of SP1 and TAFII130 disrupted in early Huntingtin's Disease.
UniProt Protein Details:

Protein type:DNA-binding; C2H2-type zinc finger protein; Transcription factor

Chromosomal Location of Human Ortholog: 12q13.1

Cellular Component: nucleoplasm; cytoplasm; nucleus

Molecular Function:protein C-terminus binding; histone acetyltransferase binding; protein binding; protein homodimerization activity; DNA binding; sequence-specific DNA binding; metal ion binding; histone deacetylase binding; double-stranded DNA binding; bHLH transcription factor binding; transcription factor binding; transcription factor activity

Biological Process: transcription initiation from RNA polymerase II promoter; embryonic placenta development; ossification; transcription, DNA-dependent; viral reproduction; megakaryocyte differentiation; positive regulation of transcription, DNA-dependent; embryonic skeletal development; embryonic process involved in female pregnancy; rhythmic process; cellular lipid metabolic process; enucleate erythrocyte differentiation; liver development; trophectodermal cell differentiation; regulation of transcription, DNA-dependent; transforming growth factor beta receptor signaling pathway; gene expression; positive regulation of transcription from RNA polymerase II promoter; embryonic camera-type eye morphogenesis; lung development

NCBI Summary:The protein encoded by this gene is a zinc finger transcription factor that binds to GC-rich motifs of many promoters. The encoded protein is involved in many cellular processes, including cell differentiation, cell growth, apoptosis, immune responses, response to DNA damage, and chromatin remodeling. Post-translational modifications such as phosphorylation, acetylation, glycosylation, and proteolytic processing significantly affect the activity of this protein, which can be an activator or a repressor. Three transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Nov 2014]
UniProt Code:P08047
NCBI GenInfo Identifier:13638437
NCBI Gene ID:6667
NCBI Accession:P08047.3
UniProt Secondary Accession:P08047,Q86TN8, Q9H3Q5, Q9NR51, Q9NY21, Q9NYE7, E4Z9M7 G5E9M8,
UniProt Related Accession:P08047
Molecular Weight:
NCBI Full Name:Transcription factor Sp1
NCBI Synonym Full Names:Sp1 transcription factor
NCBI Official Symbol:SP1  
NCBI Protein Information:transcription factor Sp1; specificity protein 1
UniProt Protein Name:Transcription factor Sp1
Protein Family:Sex-specific storage-protein
UniProt Gene Name:SP1  
UniProt Entry Name:SP1_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.Incubate the cells for overnight at 37 °C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-SP1 (Phospho-Thr453) Antibody, Anti-SP1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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