Recombinant Anti-Beta Arrestin 2 + Beta Arrestin 1 antibody [EPR22073] (ab206972)

All lanes : Anti-Beta Arrestin 2 + Beta Arrestin 1 antibody [EPR22073] (ab206972) at 1/1000 dilution

Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : Mouse brain 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: 46 kDa
Observed band size: 46-55 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The multiple bands in the brain tissue lysate are different isoform of β-Arrestin 2. All the immuno- active bands can be totally blocked by pre-incubation of antigen peptide.

All lanes : Anti-Beta Arrestin 2 + Beta Arrestin 1 antibody [EPR22073] (ab206972) at 1/1000 dilution

Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : Human lung lysate
Lane 4 : Human brain lysate
Lane 5 : Mouse brain lysate
Lane 6 : Rat brain lysate
Lane 7 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 8 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/200000 dilution

Predicted band size: 46 kDa
Observed band size: 46-55 kDa why is the actual band size different from the predicted?

Exposure time : Lane 1 - Lane 6: 10 seconds, Lane 7: 48 seconds, Lane 8: 70 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

The multiple bands in tissue lysates are different isoforms of β-Arrestin 2 based on UniProt annotation. The molecular profile/weight observed is consistent with what has been described in the literature (PMID:16820410, PMID:27759077, PMID:19955404).

Beta Arrestin 2 was immunoprecipitated from 0.35 mg of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate with ab206972 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab206972 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HEK-293T whole cell lysate 10 µg (Input). 

Lane 2: ab206972 IP in HEK-293T whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab206972 in HEK-293T whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 10 seconds.

Dot blot analysis of beta Arrestin 1+Beta Arrestin 2 labelled with ab206972 at 1/1000 dilution and 1/2000 dilution using different lot numbers (A & B), and labelled with ab213204 (His) at 1/1000 dilution (C).

Lane 1: P8423 beta Arrestin 2 peptide.
Lane 2: P8423 beta Arrestin 1 peptide.

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.
Blocking and dilution buffer: 5% NFDM/TBST.
Exposure time: 180 seconds.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling Beta Arrestin 2 with ab206972 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluorr^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.