Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: GNAQ knockout HAP1 whole cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab210004 observed at 42 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab210004 was shown to specifically react with GNAQ in wild-type HAP1 cells as signal was lost in GNAQ knockout cells. Wild-type and GNAQ knockout samples were subjected to SDS-PAGE. ab210004 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-GNAQ antibody [EPR20978] (ab210004) at 1/1000 dilution

Lane 1 : His-tagged human GNAQ (aa1-359) recombinant protein
Lane 2 : His-tagged human GNA11 (aa1-359) recombinant protein

Lysates/proteins at 0.01 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 42 kDa
Observed band size: 42 kDa


Exposure time: 1 second

Blocking and dilution buffer: 5% NFDM/TBST 

Lanes 1-4 : Anti-GNAQ antibody [EPR20978] (ab210004) at 1/2000 dilution
Lanes 5-7 : Anti-GNAQ antibody [EPR20978] (ab210004) at 1/1000 dilution

Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 3 : A549 (human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg
Lane 5 : C6 (rat glial tumor cell line) whole cell lysate at 10 µg
Lane 6 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg
Lane 7 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

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

Developed using the ECL technique.

Predicted band size: 42 kDa
Observed band size: 42 kDa

 

Exposure time : Lanes 1-3 & 5-7: 3 minutes; Lane 4: 3 seconds.

Blocking and dilution buffer: 5% NFDM/TBST

Lanes 1-4 : Anti-GNAQ antibody [EPR20978] (ab210004) at 1/2000 dilution
Lane 5 : Anti-GNAQ antibody [EPR20978] (ab210004) at 1/1000 dilution

Lane 1 : Human fetal pancreas lysate at 20 µg
Lane 2 : Mouse lung lysate at 20 µg
Lane 3 : Mouse kidney lysate at 20 µg
Lane 4 : Rat lung lysate at 20 µg
Lane 5 : Human stomach lysate at 10 µg

Secondary
Lanes 1-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 5 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Developed using the ECL technique.

Predicted band size: 42 kDa
Observed band size: 42 kDa

 

Exposure time : Lanes 2-4: 3 minutes; Lanes 1 & 5: 15 seconds.

Blocking and dilution buffer: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded human mammary gland tissue labeling GNAQ with ab210004 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and cytoplasmic staining on human mammary gland is observed (PMID: 22421440; PMID: 1946421; PMID: 18799799). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

 

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human papillary thyroid carcinoma tissue labeling GNAQ with ab210004 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and cytoplasmic staining on human mammary gland is observed (PMID: 22421440; PMID: 1946421; PMID: 18799799). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methonol-permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell line labeling GNAQ with ab210004 at 1/50 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 (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

GNAQ was immunoprecipitated from 0.35 mg of A549 (human lung carcinoma cell line) whole cell lysate with ab210004 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab210004 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: A549 whole cell lysate 10 μg (Input).

Lane 2: ab210004 IP in A549 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab210004 in A549 whole cell lysate.

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

Exposure time: 3 minutes.