Anti-GRK2 antibody [EPR22465]

• IP

Supplier Data

Immunoprecipitation - Anti-GRK2 antibody [EPR22465] (AB227825)

GRK2 was immunoprecipitated from 0.35 mg HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate with ab227825 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab227825 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.
Lane 1 : HEK-293T whole cell lysate 10 μg (Input).
Lane 2 : ab227825 IP in HEK-293T whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab227825 in HEK-293T whole cell lysate.
Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 3 seconds.

GRK2 was found readily degradable by proteolytic process (PMID : 9857063; PMID : 12738776). The bands smaller than 80-kDa detected in the immune-precipitate may represent degraded GRK2.

All lanes:

Immunoprecipitation - Anti-GRK2 antibody [EPR22465] (ab227825)

Predicted band size: 80 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-GRK2 antibody [EPR22465] (AB227825)

GRK2 was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab227825 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab227825 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.
Lane 1 : HeLa whole cell lysate 10 μg (Input).
Lane 2 : ab227825 IP in HeLa whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab227825 in HeLa whole cell lysate.
Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 3 seconds.

GRK2 was found readily degradable by proteolytic process (PMID : 9857063; PMID : 12738776). The bands smaller than 80-kDa detected in the immune-precipitate may represent degraded GRK2.

All lanes:

Immunoprecipitation - Anti-GRK2 antibody [EPR22465] (ab227825)

Predicted band size: 80 kDa

false

• WB

Lab

Western blot - Anti-GRK2 antibody [EPR22465] (AB227825)

Lanes 1-4 : Merged signal (red and green). Green - ab227825 observed at 80 kDa. Red - loading control ab8245 observed at 36 kDa.

ab227825 Anti-GRK2 antibody [EPR22465] was shown to specifically react with GRK2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266352 (knockout cell lysate ab257345) was used. Wild-type and GRK2 knockout samples were subjected to SDS-PAGE. ab227825 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-GRK2 antibody [EPR22465] (ab227825) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

ADRBK1 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human ADRBK1 (GRK2) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-adrbk1-grk2-knockout-hek-293t-cell-line-ab266352'>ab266352</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 80 kDa

Observed band size: 80 kDa

false

• WB

Supplier Data

Western blot - Anti-GRK2 antibody [EPR22465] (AB227825)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GRK2 antibody [EPR22465] (ab227825) at 1/1000 dilution

All lanes:

Human skeletal muscle lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/1000 dilution

Predicted band size: 80 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-GRK2 antibody [EPR22465] (AB227825)

Blocking/Dilution buffer : NFDM/TBST.

ab227825 was shown to specifically react with GRK2 in wild-type HAP1 cells as signal was lost in GRK2 knockout cells. Wild-type and GRK2 knockout samples were subjected to SDS-PAGE. ab227825 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.

All lanes:

Western blot - Anti-GRK2 antibody [EPR22465] (ab227825) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

GRK2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 4:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 5:

HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 80 kDa

false