Anti-RAP1/TERF2IP antibody [4c8/1]

• Flow Cyt

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Flow Cytometry - Anti-RAP1/TERF2IP antibody [4c8/1] (AB14404)

Overlay histogram showing HeLa cells stained with ab14404 (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 (ab14404, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IP

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Immunoprecipitation - Anti-RAP1/TERF2IP antibody [4c8/1] (AB14404)

RAP1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to RAP1/TERF2IP 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 ab14404.
Secondary : Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band : 55kDa; RAP1/TERF2IP

All lanes:

Immunoprecipitation - Anti-RAP1/TERF2IP antibody [4c8/1] (ab14404)

Predicted band size: 44 kDa

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• WB

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Western blot - Anti-RAP1/TERF2IP antibody [4c8/1] (AB14404)

All lanes:

Western blot - Anti-RAP1/TERF2IP antibody [4c8/1] (ab14404) at 5 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 4:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Predicted band size: 44 kDa

Observed band size: 55 kDa

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Exposure time: 8min