The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 38 kDa (predicted molecular weight: 38 kDa).
Use 5-10 µg for 25 µg of chromatin.
Use a concentration of 1 µg/ml.
Use 1µg for 106 cells.
Use at an assay dependent dilution.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
General 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.
Widely expressed, with levels highest in the testis and ovary.
Involvement in disease
Defects 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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)
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.
ChIP - Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)
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.
Western blot - Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)
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 All lanes : Rabbit polyclonal to Mouse IgG - H&L (HRP) at 1/3000 dilution
Flow Cytometry - Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)
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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)This image is courtesy of Daniel Alpern
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.
Immunocytochemistry/ Immunofluorescence - Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)
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.
Immunoprecipitation - Anti-TATA binding protein TBP antibody [mAbcam 51841] - Nuclear Loading Control and ChIP Grade (ab51841)
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.
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