Anti-PABP antibody [10E10]

• Flow Cyt

Unknown

Flow Cytometry - Anti-PABP antibody [10E10] (AB6125)

Overlay histogram showing HeLa cells stained with ab6125 (red line). The cells were fixed with 100% 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 (ab6125, 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. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• ICC

Lab

Immunocytochemistry - Anti-PABP antibody [10E10] (AB6125)

ICC/IF image of ab6125 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6125, 1µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• IP

Unknown

Immunoprecipitation - Anti-PABP antibody [10E10] (AB6125)

PABP was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to PABP (ab6125) 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 ab6125.
Secondary : Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band : 76kDa : PABP; 25kDa.

All lanes:

Immunoprecipitation - Anti-PABP antibody [10E10] (ab6125)

Predicted band size: 71 kDa

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• WB

Supplier Data

Western blot - Anti-PABP antibody [10E10] (AB6125)

Western Blot analysis on HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate using ab6125 at 1/100 to 1/6400 dilution. Anti-mouse IgG (whole molecule)-AP conjugate was used at the secondary at a 1/2000 dilution. Detection with BCIP/NBT substrate.
4-12% Bis-Tris, 1X MOPS running buffer.

All lanes:

Western blot - Anti-PABP antibody [10E10] (ab6125)

Predicted band size: 71 kDa

false