Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)

Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab51841 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
WB Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 38 kDa (predicted molecular weight: 38 kDa).
ChIP Use 5-10 µg for 25 µg of chromatin.
ICC/IF Use a concentration of 1 µg/ml.
Flow Cyt Use 1µg for 106 cells.
ChIP/Chip Use at an assay dependent dilution.
IP Use at an assay dependent concentration.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • FunctionGeneral transcription factor that functions at the core of the DNA-binding multiprotein factor TFIID. Binding of TFIID to the TATA box is the initial transcriptional step of the pre-initiation complex (PIC), playing a role in the activation of eukaryotic genes transcribed by RNA polymerase II. 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 with the rDNA promoter. SL1 is involved in stabilization of nucleolar transcription factor 1/UBTF on rDNA.
  • Tissue specificityWidely expressed, with levels highest in the testis and ovary.
  • Involvement in diseaseDefects in TBP are the cause of spinocerebellar ataxia type 17 (SCA17) [MIM:607136]. Spinocerebellar ataxia is a clinically and genetically heterogeneous group of cerebellar disorders. Patients show progressive incoordination of gait and often poor coordination of hands, speech and eye movements, due to degeneration of the cerebellum with variable involvement of the brainstem and spinal cord. SCA17 is an autosomal dominant cerebellar ataxia (ADCA) characterized by widespread cerebral and cerebellar atrophy, dementia and extrapyramidal signs. The molecular defect in SCA17 is the expansion of a CAG repeat in the coding region of TBP. Longer expansions result in earlier onset and more severe clinical manifestations of the disease.
  • Sequence similaritiesBelongs to the TBP family.
  • Cellular localizationNucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • GTF2D antibody
    • GTF2D1 antibody
    • HDL4 antibody
    • MGC117320 antibody
    • MGC126054 antibody
    • MGC126055 antibody
    • SCA17 antibody
    • TATA binding factor antibody
    • TATA box factor antibody
    • TATA sequence binding protein antibody
    • TATA sequence-binding protein antibody
    • TATA-binding factor antibody
    • TATA-box binding protein N-terminal domain antibody
    • TATA-box factor antibody
    • TATA-box-binding protein antibody
    • TBP antibody
    • TBP_HUMAN antibody
    • TFIID antibody
    • Transcription initiation factor TFIID TBP subunit antibody
    see all

Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade images

  • IHC image of TATA binding protein TBP staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab51841, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 5 µg of  ab51841 (blue), and 20 µl 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 or sybr green approach). Primers and probes are located within 1 kb of the transcription start site. 

  • All lanes : Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841) at 5 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Cytoplasmic Lysate at 10 µg
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg
    Lane 3 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 20 µg

    Secondary
    Rabbit polyclonal to Mouse IgG - H&L (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size : 38 kDa
    Observed band size : 40 kDa (why is the actual band size different from the predicted?)
  • Overlay histogram showing HeLa cells stained with ab51841 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab51841, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • ab51841 staining TATA binding protein TBP and ab15102 staining Claudin3 in Mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 10% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer pH6. Samples were incubated with primary antibodies ab51841 and ab15102 (1/400 and 1/300 respectively in blocking buffer) for 16 hours at 4°C. A Cy3-conjugated Goat anti-mouse IgG polyclonal (1/200) was used as the secondary antibody.

     

    See Abreview

  • ICC/IF image of ab51841 stained human HEK 293 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab51841, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa, HepG2 and MCF7 cells.

  • TBP was immunoprecipitated using 0.5mg Hela whole cell extract, 10µg of Mouse monoclonal to TBP and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab51841.
    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

    Band: 40kDa: TATA binding protein TBP.

References for Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)

This product has been referenced in:
  • Martianov I  et al. TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression. Sci Rep 6:32069 (2016). Mouse . Read more (PubMed: 27576952) »
  • Cattoglio C  et al. Functional and mechanistic studies of XPC DNA-repair complex as transcriptional coactivator in embryonic stem cells. Proc Natl Acad Sci U S A 112:E2317-26 (2015). Read more (PubMed: 25901318) »

See all 24 Publications for this product

Product Wall

Application ChIP
Sample Human 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
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Submitted Sep 15 2015

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Sample Mouse Cell (MS)
Specification MS
Permeabilization Yes - Triton 1%
Fixative Formaldehyde
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Submitted Feb 25 2015

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Mouse Cell lysate - whole cell (brain)
Specification brain
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Aug 22 2014

Application Immunoprecipitation
Immuno-precipitation step Other - Magnetic beads
Sample Mouse Cell lysate - nuclear (mES)
Specification mES
Total protein in input 500 µg
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Submitted May 13 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step FBS / BSA as blocking agent for 4 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Tris pH 9
Sample Mouse Tissue sections (Embryo 15.5 dpc)
Specification Embryo 15.5 dpc
Permeabilization Yes - Tween 20, 0,1%
Fixative Formaldehyde
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Submitted Feb 25 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (4-20%)
Sample Mouse Tissue lysate - nuclear (Liver)
Specification Liver
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Jan 22 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Heat-inactivated normal donkey serum in 0.05% PBS-T as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Antigen retrieval step None
Sample Mouse Tissue sections (Mouse, whole brain sections)
Specification Mouse, whole brain sections
Permeabilization Yes - Tween-20
Fixative Paraformaldehyde
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Submitted Jan 13 2014



Si se pretende almacenar el anticuerpo durante largos periodos (más de una o dos semanas), lo ideal es alicuotarlo en volúmenes de trabajo, y congelarlo a -20 ºC o -80 ºC, evitando siempre ciclos de congelación ...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Human Cell lysate - whole cell (293T cells)
Specification 293T cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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Submitted Sep 23 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 30 µg
Gel Running Conditions Non-reduced Denaturing (12%)
Sample Mouse Cell lysate - whole cell (primary hepatoctyes)
Specification primary hepatoctyes
Treatment 1ug/ml Doxo 24hrs
Blocking step I-Block(Applied biosystems) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2µg/mL · Temperature: 22°C
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Submitted Jul 08 2013

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"