All lanes : Anti-eRF3/GSPT1 + eRF3B/GSPT2 antibody [EPR22908-63] (ab256471) at 1/1000 dilution

Lane 1 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 56 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Exposure time: 3 minutes.

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST.

All lanes : Anti-eRF3/GSPT1 + eRF3B/GSPT2 antibody [EPR22908-63] (ab256471) at 1/1000 dilution

Lane 1 : His tagged human eRF3a recombinant protein 10 ng
Lane 2 : His tagged human eRF3b recombinant protein 10 ng

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 56 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

The loading samples are E.coil extracts containing eRF3a or eRF3b recombinant protein respectively.

Exposure time: 3 minutes.

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST.

All lanes : Anti-eRF3/GSPT1 + eRF3B/GSPT2 antibody [EPR22908-63] (ab256471) at 1/1000 dilution

Lane 1 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : Rat kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 56 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Exposure time: Lane 1: 48 seconds, Lane 2: 15 seconds, Lane 3: 48 seconds.

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST.

eRF3a+eRF3b was immunoprecipitated from 0.35 mg PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate with ab256471 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256471. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate 10µg

Lane 2: ab256471 IP in PC-3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256471 in PC-3 whole cell lysate.

Blocking and dilution buffer and concentration/ 5% NFDM/TBST.

Exposure time: 3 seconds.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling eRF3a+eRF3b with ab256471 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling eRF3a+eRF3b with ab256471 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling eRF3a+eRF3b with ab256471 at 1/1000 (5 ug/ml) dilution, followed by anti- eRF3a+eRF3b ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling eRF3a+eRF3b with ab256471 at 1/1000 (5 ug/ml) dilution, followed by anti- eRF3a+eRF3b ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in HeLa cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control/ Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.