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TAF5L Colorimetric Cell-Based ELISA

SKU:
CBCAB00953
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Detection Method:
Colorimetric
$719
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Description

system_update_altDatasheetMSDS

TAF5L Colorimetric Cell-Based ELISA

The TAF5L Colorimetric Cell-Based ELISA Kit is specifically designed for researchers looking to analyze TAF5L levels in cell lysates and supernatants. With its high sensitivity and specificity, this kit provides accurate and reproducible results, making it an ideal tool for a variety of cell-based research applications.TAF5L is a key component of the TFIID complex, essential for the initiation of transcription in eukaryotic cells. Dysregulation of TAF5L has been implicated in various diseases, including cancer and developmental disorders, making it a valuable target for further study and potential therapeutic interventions.

With the TAF5L Colorimetric Cell-Based ELISA Kit, researchers can confidently measure TAF5L levels in their cell samples, gaining valuable insights into the role of this protein in cellular processes and disease pathology. Order your kit today to advance your research in this important area of study.

Product Name:TAF5L Colorimetric Cell-Based ELISA
Product Code:CBCAB00953
ELISA Type:Cell-Based
Target:TAF5L
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The TAF5L Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TAF5L protein expression profile in cells. The kit can be used for measuring the relative amounts of TAF5L in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TAF5L.

Qualitative determination of TAF5L concentration is achieved by an indirect ELISA format. In essence, TAF5L is captured by TAF5L-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: 27097, UniProt ID: O75529, OMIM: 0, Unigene: Hs.270621
Gene Symbol:TAF5L
Sub Type:None
UniProt Protein Function:TAF5L: Functions as a component of the PCAF complex. The PCAF complex is capable of efficiently acetylating histones in a nucleosomal context. The PCAF complex could be considered as the human version of the yeast SAGA complex. Belongs to the WD repeat TAF5 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Transcription factor

Chromosomal Location of Human Ortholog: 1q42.13

Molecular Function:histone acetyltransferase activity; protein binding; transcription coactivator activity; transcription factor activity

Biological Process: establishment and/or maintenance of chromatin architecture; regulation of transcription, DNA-dependent; transcription from RNA polymerase II promoter

NCBI Summary:The product of this gene belongs to the WD-repeat TAF5 family of proteins. This gene encodes a protein that is a component of the PCAF histone acetylase complex. The PCAF histone acetylase complex, which is composed of more than 20 polypeptides some of which are TAFs, is required for myogenic transcription and differentiation. TAFs may participate in basal transcription, serve as coactivators, function in promoter recognition or modify general transcription factors to facilitate complex assembly and transcription initiation. The encoded protein is structurally similar to one of the histone-like TAFs, TAF5. Alternatively spliced transcript variants encoding different isoforms have been identified for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:O75529
NCBI GenInfo Identifier:46577305
NCBI Gene ID:27097
NCBI Accession:O75529.1
UniProt Secondary Accession:O75529,Q5TDI5, Q5TDI6, Q8IW31,
UniProt Related Accession:O75529
Molecular Weight:36,991 Da
NCBI Full Name:TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 5L
NCBI Synonym Full Names:TATA-box binding protein associated factor 5 like
NCBI Official Symbol:TAF5L  
NCBI Official Synonym Symbols:PAF65B  
NCBI Protein Information:TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 5L
UniProt Protein Name:TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 5L
UniProt Synonym Protein Names:PCAF-associated factor 65 beta; PAF65-beta
Protein Family:TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor
UniProt Gene Name:TAF5L  
UniProt Entry Name:TAF5L_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-TAF5L 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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