Human TAF1 (TATA Box Binding Protein Associated Factor 1) ELISA Kit (HUES02069)

SKU:
HUES02069
€539

Description

ELISA Kit Technical ManualMSDS

Human TAF1 (TATA Box Binding Protein Associated Factor 1) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human TAF1 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human TAF1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human TAF1, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The OD value is proportional to the concentration of Human TAF1. The concentration of Human TAF1 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range15.63-1000 pg/mL
Sensitivity9.38 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human TAF1 in samples. No significant cross-reactivity or interference between Human TAF1 and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(pg/mL)
O.D Average Corrected
10002.414
2.454
2.4342.353
5001.564
1.608
1.5861.505
2500.962
0.924
0.9430.862
1250.518
0.534
0.5260.445
62.50.258
0.252
0.2550.174
31.250.191
0.173
0.1820.101
15.630.125
0.141
0.1330.052
00.076
0.086
0.081--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TAF1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TAF1 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)48.44143.68352.8945.90129.57377.21
Standard deviation3.368.3217.642.545.9618.71
C V (%)6.945.795.005.534.604.96

Recovery

The recovery of Human TAF1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)94-106101
EDTA plasma (n=5)89-10496
Cell culture media (n=5)89-10296

Linearity

Samples were spiked with high concentrations of Human TAF1 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)86-9786-9992-105
Average (%)929298
1:4Range (%)94-10884-9685-98
Average (%)999090
1:8Range (%)90-10682-9486-99
Average (%)978792
1:16Range (%)89-10485-9685-97
Average (%)969090

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human TAF1 (TATA Box Binding Protein Associated Factor 1) ELISA Kit (HUES02069) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37 °C.
    7. Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human TAF1 (TATA Box Binding Protein Associated Factor 1) ELISA Kit (HUES02069) Protein Information

UniProt Protein Function:TAF1: Largest component and core scaffold of the TFIID basal transcription factor complex. Contains novel N- and C-terminal Ser/Thr kinase domains which can autophosphorylate or transphosphorylate other transcription factors. Phosphorylates TP53 on 'Thr-55' which leads to MDM2-mediated degradation of TP53. Phosphorylates GTF2A1 and GTF2F1 on Ser residues. Possesses DNA- binding activity. Essential for progression of the G1 phase of the cell cycle. Defects in TAF1 are the cause of dystonia type 3 (DYT3); also called X-linked dystonia-parkinsonism (XDP). DYT3 is a X-linked dystonia-parkinsonism disorder. Dystonia is defined by the presence of sustained involuntary muscle contractions, often leading to abnormal postures. DYT3 is characterized by severe progressive torsion dystonia followed by parkinsonism. Its prevalence is high in the Philippines. DYT3 has a well-defined pathology of extensive neuronal loss and mosaic gliosis in the striatum (caudate nucleus and putamen) which appears to resemble that in Huntington disease. Belongs to the TAF1 family. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Kinase, protein; Protein kinase, atypical; EC 2.7.11.1; DNA-binding; Transcription, coactivator/corepressor; EC 2.3.1.48; Protein kinase, Ser/Thr (non-receptor); ATYPICAL group; TAF1 family

Chromosomal Location of Human Ortholog: Xq13.1

Cellular Component: nucleoplasm; transcription factor TFIID complex

Molecular Function:histone acetyltransferase activity; p53 binding; protein binding; protein serine/threonine kinase activity; sequence-specific DNA binding; TATA-binding protein binding; transcription coactivator activity; transcription factor binding

Biological Process: peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; positive regulation of transcription from RNA polymerase II promoter; protein amino acid autophosphorylation; response to DNA damage stimulus; RNA elongation from RNA polymerase II promoter; transcription from RNA polymerase II promoter; transcription initiation; transcription initiation from RNA polymerase II promoter; transcriptional preinitiation complex assembly

Disease: Dystonia 3, Torsion, X-linked

NCBI Summary:Initiation of transcription by RNA polymerase II requires the activities of more than 70 polypeptides. The protein that coordinates these activities is the basal transcription factor TFIID, which binds to the core promoter to position the polymerase properly, serves as the scaffold for assembly of the remainder of the transcription complex, and acts as a channel for regulatory signals. TFIID is composed of the TATA-binding protein (TBP) and a group of evolutionarily conserved proteins known as TBP-associated factors or TAFs. TAFs may participate in basal transcription, serve as coactivators, function in promoter recognition or modify general transcription factors (GTFs) to facilitate complex assembly and transcription initiation. This gene encodes the largest subunit of TFIID. This subunit binds to core promoter sequences encompassing the transcription start site. It also binds to activators and other transcriptional regulators, and these interactions affect the rate of transcription initiation. This subunit contains two independent protein kinase domains at the N- and C-terminals, but also possesses acetyltransferase activity and can act as a ubiquitin-activating/conjugating enzyme. Mutations in this gene result in Dystonia 3, torsion, X-linked, a dystonia-parkinsonism disorder. Alternative splicing of this gene results in multiple transcript variants. This gene is part of a complex transcription unit (TAF1/DYT3), wherein some transcript variants share exons with TAF1 as well as additional downstream DYT3 exons. [provided by RefSeq, Oct 2013]
UniProt Code:P21675
NCBI GenInfo Identifier:115942
NCBI Gene ID:6872
NCBI Accession:P21675.2
UniProt Secondary Accession:P21675,Q59FZ3, Q6IUZ1, Q70Q86, Q70Q87, Q70T00, Q70T01 A5CVC8, A5CVC9, A5CVD0, A5CVD1, B1Q2X3,
UniProt Related Accession:P21675
Molecular Weight:214,913 Da
NCBI Full Name:Transcription initiation factor TFIID subunit 1
NCBI Synonym Full Names:TATA-box binding protein associated factor 1
NCBI Official Symbol:TAF1
NCBI Official Synonym Symbols:OF; XDP; BA2R; CCG1; CCGS; DYT3; KAT4; P250; NSCL2; TAF2A; MRXS33; N-TAF1; TAFII250; DYT3/TAF1; TAFII-250; TAF(II)250
NCBI Protein Information:transcription initiation factor TFIID subunit 1
UniProt Protein Name:Transcription initiation factor TFIID subunit 1
UniProt Synonym Protein Names:Cell cycle gene 1 protein; TBP-associated factor 250 kDa; p250; Transcription initiation factor TFIID 250 kDa subunit; TAF(II)250; TAFII-250; TAFII250
Protein Family:Taz1-interacting factor
UniProt Gene Name:TAF1
UniProt Entry Name:TAF1_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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