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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human TATA binding protein TBP.
(Peptide available as ab25711.)
Alternative versions available:
Anti-TATA binding protein TBP antibody (Alexa Fluor® 647) [mAbcam 51841] (ab197873)
Anti-TATA binding protein TBP antibody (Alexa Fluor® 488) [mAbcam 51841] (ab197872)
Anti-TATA binding protein TBP antibody (HRP) [mAbcam 51841] (ab197874)
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.
|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.|
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.
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.
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.