USF2 Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00898
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
Frequently bought together:

Description

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USF2 Colorimetric Cell-Based ELISA Kit

The USF2 Colorimetric Cell-Based ELISA Kit is specifically designed for the precise measurement of USF2 levels in cell lysates and tissue extracts. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for your research needs.USF2, also known as upstream transcription factor 2, plays a critical role in the regulation of gene expression and is involved in various cellular processes such as cell proliferation, differentiation, and apoptosis.

Aberrant USF2 expression has been linked to various diseases including cancer, diabetes, and cardiovascular disorders, making it a valuable marker for studying disease mechanisms and potential therapeutic interventions.With its user-friendly protocol and reliable performance, the USF2 Colorimetric Cell-Based ELISA Kit is an indispensable tool for researchers looking to delve deeper into the role of USF2 in cellular pathways and disease pathology.

Product Name:USF2 Colorimetric Cell-Based ELISA
Product Code:CBCAB00898
ELISA Type:Cell-Based
Target:USF2
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The USF2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect USF2 protein expression profile in cells. The kit can be used for measuring the relative amounts of USF2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on USF2.

Qualitative determination of USF2 concentration is achieved by an indirect ELISA format. In essence, USF2 is captured by USF2-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 7392, UniProt ID: Q15853, OMIM: 600390, Unigene: Hs.454534
Gene Symbol:USF2
Sub Type:None
UniProt Protein Function:USF2: Transcription factor that binds to a symmetrical DNA sequence (E-boxes) (5'-CACGTG-3') that is found in a variety of viral and cellular promoters. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:DNA-binding

Chromosomal Location of Human Ortholog: 19q13

Cellular Component: intracellular membrane-bound organelle; nucleoplasm; nucleus

Molecular Function:bHLH transcription factor binding; protein binding; protein heterodimerization activity; protein homodimerization activity; RNA polymerase II transcription factor activity, enhancer binding; sequence-specific DNA binding; transcription factor activity

Biological Process: late viral mRNA transcription; lipid homeostasis; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription from RNA polymerase II promoter by glucose; regulation of transcription from RNA polymerase II promoter by glucose; transcription from RNA polymerase II promoter

NCBI Summary:This gene encodes a member of the basic helix-loop-helix leucine zipper family of transcription factors. The encoded protein can activate transcription through pyrimidine-rich initiator (Inr) elements and E-box motifs and is involved in regulating multiple cellular processes. [provided by RefSeq, Mar 2016]
UniProt Code:Q15853
NCBI GenInfo Identifier:2833271
NCBI Gene ID:7392
NCBI Accession:Q15853.1
UniProt Secondary Accession:Q15853,O00671, O00709, Q05750, Q07952, Q15851, Q15852 Q6FI33, Q6YI47,
UniProt Related Accession:Q15853
Molecular Weight:23,988 Da
NCBI Full Name:Upstream stimulatory factor 2
NCBI Synonym Full Names:upstream transcription factor 2, c-fos interacting
NCBI Official Symbol:USF2  
NCBI Official Synonym Symbols:FIP; bHLHb12  
NCBI Protein Information:upstream stimulatory factor 2
UniProt Protein Name:Upstream stimulatory factor 2
UniProt Synonym Protein Names:Class B basic helix-loop-helix protein 12; bHLHb12; FOS-interacting protein; FIP; Major late transcription factor 2; Upstream transcription factor 2
Protein Family:Upstream stimulatory factor
UniProt Gene Name:USF2  
UniProt Entry Name:USF2_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-USF2 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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