All lanes : Anti-EEF1A2 antibody [EPR22651-46] (ab227824) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : EEF1A2 knockout HAP1 whole cell lysate
Lane 3 : HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 4 : Neuro-2a (mouse neuroblastoma neuroblast), 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: 50 kDa
Observed band size: 50 kDa

ab227824 was shown to specifically react with EEF1A2 in wild-type HAP1 cells as signal was lost in EEF1A2 knockout cells. Wild-type and EEF1A2 knockout samples were subjected to SDS-PAGE. ab227824 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

This antibody also recognizes a weakly unidentifiable band at around 75kDa

Blocking/diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 3.25 seconds

Anti-EEF1A2 antibody [EPR22651-46] (ab227824) at 1/1000 dilution + Human brain tissue lysate at 20 µg

Secondary
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 50 kDa
Observed band size: 50 kDa

Blocking/diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 7.75 seconds

All lanes : Anti-EEF1A2 antibody [EPR22651-46] (ab227824) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart tissue lysate
Lane 3 : Rat brain tissue lysate
Lane 4 : Rat heart 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: 50 kDa
Observed band size: 50 kDa

Blocking/diluting buffer and concentration: 5% NFDM/TBST

Exposure Time: 3.25 seconds

EEF1A2 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) whole cell lysate with ab227824 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using Ab227824 1/1000 dilution (0.54 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: HEK-293T (human embryonic kidney epithelial cell) whole cell lysate 10μg

Lane 2: ab227824 IP in HEK-293T whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227824 in HEK-293T whole cell lysate.

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

Exposure time: 15 seconds