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TAF1A Colorimetric Cell-Based ELISA

SKU:
CBCAB01109
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

TAF1A Colorimetric Cell-Based ELISA

The TAF1A Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the detection of TAF1A protein levels in cell lysates and tissue samples. This kit offers high sensitivity and specificity, providing researchers with accurate and reliable results for their experiments.TAF1A is a key protein involved in transcriptional regulation and DNA repair processes, making it a crucial target for studying gene expression dynamics and cellular responses to various stimuli. It is known to be dysregulated in certain diseases, including cancer and genetic disorders, highlighting its importance as a potential biomarker for disease diagnosis and treatment.

With the TAF1A Colorimetric Cell-Based ELISA Kit, researchers can easily quantify TAF1A protein levels in their samples, enabling them to gain valuable insights into the molecular mechanisms underlying disease pathology and identify potential therapeutic targets. This kit is a valuable tool for advancing research in the fields of molecular biology, genetics, and oncology.

Product Name:TAF1A Colorimetric Cell-Based ELISA
Product Code:CBCAB01109
ELISA Type:Cell-Based
Target:TAF1A
Reactivity:Human, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The TAF1A Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TAF1A protein expression profile in cells. The kit can be used for measuring the relative amounts of TAF1A in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TAF1A.

Qualitative determination of TAF1A concentration is achieved by an indirect ELISA format. In essence, TAF1A is captured by TAF1A-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: 9015, UniProt ID: Q15573, OMIM: 604903, Unigene: Hs.153088
Gene Symbol:TAF1A
Sub Type:None
UniProt Protein Function:TAF1A: Component of the transcription factor SL1/TIF-IB complex, which is involved in the assembly of the PIC (preinitiation complex) during RNA polymerase I-dependent transcription. The rate of PIC formation probably is primarily dependent on the rate of association of SL1/TIF-IB with the rDNA promoter. SL1/TIF-IB is involved in stabilization of nucleolar transcription factor 1/UBTF on rDNA. Formation of SL1/TIF-IB excludes the association of TBP with TFIID subunits. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Chromosomal Location of Human Ortholog: 1q42

Cellular Component: intracellular membrane-bound organelle; microtubule cytoskeleton; nucleoplasm; nucleus; RNA polymerase I transcription factor complex

Molecular Function:protein binding

Biological Process: positive regulation of gene expression, epigenetic; RNA elongation from RNA polymerase I promoter; termination of RNA polymerase I transcription; transcription from RNA polymerase I promoter; transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase I promoter

NCBI Summary:This gene encodes a subunit of the RNA polymerase I complex, Selectivity Factor I (SLI). The encoded protein is a TATA box-binding protein-associated factor that plays a role in the assembly of the RNA polymerase I preinitiation complex. Alternate splicing results in multiple transcript variants encoding multiple isoforms.[provided by RefSeq, Jan 2011]
UniProt Code:Q15573
NCBI GenInfo Identifier:74739864
NCBI Gene ID:9015
NCBI Accession:Q15573.1
UniProt Secondary Accession:Q15573,Q9NWA1, B2RDZ8, D3DTB7,
UniProt Related Accession:Q15573
Molecular Weight:39,615 Da
NCBI Full Name:TATA box-binding protein-associated factor RNA polymerase I subunit A
NCBI Synonym Full Names:TATA-box binding protein associated factor, RNA polymerase I subunit A
NCBI Official Symbol:TAF1A  
NCBI Official Synonym Symbols:SL1; RAFI48; TAFI48; MGC:17061  
NCBI Protein Information:TATA box-binding protein-associated factor RNA polymerase I subunit A
UniProt Protein Name:TATA box-binding protein-associated factor RNA polymerase I subunit A
UniProt Synonym Protein Names:RNA polymerase I-specific TBP-associated factor 48 kDa; TAFI48; TATA box-binding protein-associated factor 1A; TBP-associated factor 1A; Transcription factor SL1; Transcription initiation factor SL1/TIF-IB subunit A
Protein Family:TATA box-binding protein-associated factor
UniProt Gene Name:TAF1A  
UniProt Entry Name:TAF1A_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-TAF1A 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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