Overview

  • Product name
    Anti-KAT4 / TBP Associated Factor 1 antibody
    See all KAT4 / TBP Associated Factor 1 primary antibodies
  • Description
    Rabbit polyclonal to KAT4 / TBP Associated Factor 1
  • Host species
    Rabbit
  • Specificity
    ab28450 recognises transcription initiation factor TFIID subunit 1 (TAF1).
  • Tested applications
    Suitable for: IHC-P, ELISA, WB, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat, Dog
  • Immunogen

    Synthetic peptide within Human KAT4/ TBP Associated Factor 1 aa 1823-1872 (C terminal). The exact sequence is proprietary.
    Sequence:

    YEVSEEEEDEEEEEQRSGPSVLSQVHLSEDEEDSEDFHSIAGDSDLDSDE


    Database link: P21675

  • Positive control
    • Daudi cell lysate, Human kidney tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab28450 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use at an assay dependent concentration.
ELISA 1/62500.
WB Use a concentration of 0.5 µg/ml. Predicted molecular weight: 212 kDa. Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.
ChIP Use 5-10 µg for 25 µg of chromatin.

Target

  • Function
    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.
  • Involvement in disease
    Defects in TAF1 are the cause of dystonia type 3 (DYT3) [MIM:314250]; 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.
  • Sequence similarities
    Belongs to the TAF1 family.
    Contains 2 bromo domains.
    Contains 1 HMG box DNA-binding domain.
    Contains 2 protein kinase domains.
  • Post-translational
    modifications
    Phosphorylated by casein kinase II in vitro.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • BA2R antibody
    • CCG1 antibody
    • CCGS antibody
    • Cell cycle G1 phase defect antibody
    • Cell cycle gene 1 protein antibody
    • Complementation of cell cycle block G1 to S antibody
    • DYT3 antibody
    • N TAF1 antibody
    • NSCL2 antibody
    • OF antibody
    • p250 antibody
    • TAF 1 antibody
    • TAF(II)250 antibody
    • TAF1 antibody
    • TAF1 RNA polymerase II TATA box binding protein (TBP) associated factor 250kDa antibody
    • TAF1_HUMAN antibody
    • TAF2A antibody
    • TAFII-250 antibody
    • TAFII250 antibody
    • TATA box binding protein (TBP) associated factor RNA polymerase II A 250kD antibody
    • TBP associated factor 250 kDa antibody
    • TBP-associated factor 250 kDa antibody
    • Transcription factor TFIID p250 polypeptide antibody
    • Transcription initiation factor TFIID 250 kDa subunit antibody
    • Transcription initiation factor TFIID subunit 1 antibody
    • XDP antibody
    see all

Images

  • Anti-KAT4 / TBP Associated Factor 1 antibody (ab28450) at 0.5 µg/ml + Daudi cell lysate

    Predicted band size: 212 kDa

  • Quiescent human colon carcinoma HCT116 cultures were treated with 10% FBS for three time points (0, 15, 30 minutes) or (0, 30, 60 minutes) were used in Matrix-ChIP and real-time PCR assays at EGR1 gene (Exon1) and 15kb upstream site.

  • This image shows human kidney stained with ab28450 at 8µg/ml. Magnification 400x.
  • Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10mins. The  ChIP was performed with 25µg of chromatin, 8µg of  ab28450 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman and sybr green approach). Primers and probes for the actively transcribed genes GAPDH and ACT1 are located in the core promoter.
       

References

This product has been referenced in:
  • Reeder JE  et al. HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells. Elife 4:N/A (2015). ChIP . Read more (PubMed: 26488441) »
  • Iwata J  et al. Transforming growth factor-beta regulates basal transcriptional regulatory machinery to control cell proliferation and differentiation in cranial neural crest-derived osteoprogenitor cells. J Biol Chem 285:4975-82 (2010). WB ; Mouse . Read more (PubMed: 19959467) »
See all 3 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Application
ChIP
Sample
Mouse Cell lysate - nuclear (ESCs)
Specification
ESCs
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde 1 % (37ºC)
Positive control
samples

Abcam user community

Verified customer

Submitted Sep 15 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Human fibromatosis)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris EDTA pH 9
Permeabilization
Yes - Tween-20
Specification
Human fibromatosis
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Apr 15 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris EDTA pH 9
Sample
Dog Tissue sections (MDCK pellets in paraffin)
Specification
MDCK pellets in paraffin
Permeabilization
Yes - Tween-20
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Mar 04 2015

Question

BATCH NUMBER 253830 ORDER NUMBER 209721 DESCRIPTION OF THE PROBLEM No detectable enrichment at GAPDH or c-myc promoters in ChIP SAMPLE human PC3 cell line (prostate epithelial) PRIMARY ANTIBODY none DETECTION METHOD qPCR is performed by 32P-dCTP incorporation. POSITIVE AND NEGATIVE CONTROLS USED Negative control is a ChIP performed without antibody to control for background DNA recovery. ANTIBODY STORAGE CONDITIONS I used this antibody the day it arrived right out of the box, remaining antibody was aliquoted and stored at -80C. SAMPLE PREPARATION ChIP was performed on fragmented chromatin from ~10^7 cells in 1ml 20mM Tris pH 8.0, 150mM NaCl, 2mM EDTA, 1% Triton X-100 + 1X Complete Protease Inhibitor (Roche). CROSSLINKING Cells were cross linked with 1% formaldehyde for 5min at room temp. DNA FRAGMENTATION Chromatin was fragmented by 10 rounds of sonication in 30sec bursts. IP STEP 5ug of antibody was added to chromatin, tubes were rotated 3hr at 4C, Protein A Sepharose beads were added to capture immunocomplexes and tubes were rotated overnight at 4C. Beads were washed sequentially with 3 wash buffers followed by 2 washes in TE (all containing protease inhibitors) DECROSSLINKING Proteinase K (20ug, RNA-grade) digestion is performed in TE + 0.5% SDS at 55C for at least 1hr. NaCl is added to 250mM, tubes are placed at 65C 4hr-overnight to reverse crosslinking. DNA PURIFICATION DNA is purified by Phenol/Chloroform/Isoamyl Alcohol extraction followed by Ethanol precipitation. PELLET AFTER PRECIPITATION DNA is precipitated in the presence of GlycoBlue, and pellets are clearly visible. PRIMERS TESTED ON GENOMIC/INPUT DNA Primers are used to amplify both Input DNA and ChIP DNA in the PCR run. NO-TEMPLATE CONTROL IN PCR no HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. This antibody has been applied by Abcams chromatin postdoc scientist by adding 5-10µg of antibody to 25µg of chromatin. Please can you tell me the mass of chromatin that you have been using in relation to the mas of chromatin; is this determined? I was interested in the duration of fixation that you have been using. in my opinion a 5 minute duration is a little short, a duration that is suitable for the chromatin immunoprecipitation of histone modifications but not chromatin associated (transcription) factors. We commonly use a 10 minute duration of formaldehyde fixation. Our XChIP lab protocol can be located at the following link; www.abcam.com/chip I would also like to know the region of GAPDH that you are amplifying; the primers that my colleague uses amplifies the core promoter region of the GAPDH gene. I look forward to hearing from you.

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