All lanes : Anti-TATA binding protein TBP antibody [mAbcam 51841] - 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

Performed under reducing conditions.

Predicted band size: 38 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

IHC image of TATA binding protein TBP staining in human breast carcinoma FFPE section, performed on a Bond^TM 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. 

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.

 

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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 70^oC; 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.