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TF3C2 Colorimetric Cell-Based ELISA

SKU:
CBCAB01164
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheet

TF3C2 Colorimetric Cell-Based ELISA

The TF3C2 Colorimetric Cell-Based ELISA Kit is a comprehensive tool designed for the accurate detection of transcription factor 3C2 (TF3C2) levels in cell cultures. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.TF3C2 is a key transcription factor involved in the regulation of gene expression, playing a critical role in cellular functions and development. Studying TF3C2 levels can provide valuable insights into various biological processes, making it an essential biomarker for researchers investigating gene regulation and cellular signaling pathways.

The TF3C2 Colorimetric Cell-Based ELISA Kit is easy to use and provides rapid results, allowing researchers to efficiently analyze TF3C2 levels in cell samples. With its high performance and accuracy, this kit is an invaluable tool for advancing research in molecular biology, cell biology, and related fields.

Product Name:TF3C2 Colorimetric Cell-Based ELISA
Product Code:CBCAB01164
ELISA Type:Cell-Based
Target:TF3C2
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The TF3C2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TF3C2 protein expression profile in cells. The kit can be used for measuring the relative amounts of TF3C2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TF3C2.

Qualitative determination of TF3C2 concentration is achieved by an indirect ELISA format. In essence, TF3C2 is captured by TF3C2-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: 2976, UniProt ID: Q8WUA4, OMIM: 604883, Unigene: Hs.75782
Gene Symbol:TF3C2
Sub Type:None
UniProt Protein Function:GTF3C2: Required for RNA polymerase III-mediated transcription. Component of TFIIIC that initiates transcription complex assembly on tRNA and is required for transcription of 5S rRNA and other stable nuclear and cytoplasmic RNAs. May play a direct role in stabilizing interactions of TFIIIC2 with TFIIIC1. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Transcription initiation complex

Chromosomal Location of Human Ortholog: 2p23.3

Cellular Component: nucleoplasm; transcription factor TFIIIC complex

Molecular Function:DNA binding

Biological Process: 5S class rRNA transcription; gene expression; transcription from RNA polymerase III promoter; transcription, DNA-dependent; tRNA transcription from RNA polymerase III promoter

UniProt Code:Q8WUA4
NCBI GenInfo Identifier:48428661
NCBI Gene ID:2976
NCBI Accession:Q8WUA4.2
UniProt Secondary Accession:Q8WUA4,Q16632, Q9BWI7, D6W557,
UniProt Related Accession:Q8WUA4
Molecular Weight:64,003 Da
NCBI Full Name:General transcription factor 3C polypeptide 2
NCBI Synonym Full Names:general transcription factor IIIC subunit 2
NCBI Official Symbol:GTF3C2  
NCBI Official Synonym Symbols:TFIIIC110; TFIIIC-BETA  
NCBI Protein Information:general transcription factor 3C polypeptide 2
UniProt Protein Name:General transcription factor 3C polypeptide 2
UniProt Synonym Protein Names:TF3C-beta; Transcription factor IIIC 110 kDa subunit; TFIIIC 110 kDa subunit; TFIIIC110; Transcription factor IIIC subunit beta
Protein Family:General transcription factor 3C polypeptide
UniProt Gene Name:GTF3C2  
UniProt Entry Name:TF3C2_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-TF3C2 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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