Anti-A-Raf antibody [EPR16208]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-A-Raf antibody [EPR16208] (AB200653)

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling A-Raf with ab200653 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Cytoplasm staining on Human cervix carcinoma tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-A-Raf antibody [EPR16208] (AB200653)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling A-Raf with ab200653 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Cytoplasm staining on HeLa cell line is observed.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
-ve control 1 : ab200653 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-A-Raf antibody [EPR16208] (AB200653)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK293 (Human embryonic kidney) cells labeling A-Raf with ab200653 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Cytoplasm staining on HEK293 cell line is observed.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
-ve control 1 : ab200653 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-A-Raf antibody [EPR16208] (AB200653)

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling A-Raf with ab200653 at 1/100 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-A-Raf antibody [EPR16208] (AB200653)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling A-Raf with ab200653 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Cytoplasm staining on Human kidney tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

• WB

Supplier Data

Western blot - Anti-A-Raf antibody [EPR16208] (AB200653)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-A-Raf antibody [EPR16208] (ab200653) at 1/1000 dilution

Lane 1:

HT-29 (Human colorectal adenocarcinoma cells) whole cell lysate at 10 µg

Lane 2:

A375 (Human malignant melanoma) whole cell lysate at 10 µg

Lane 3:

Human fetal heart lysate at 10 µg

Lane 4:

Human fetal kidney lysate at 10 µg

Lane 5:

Human bladder lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 68 kDa

Observed band size: 68 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-A-Raf antibody [EPR16208] (AB200653)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-A-Raf antibody [EPR16208] (ab200653) at 1/1000 dilution

All lanes:

Human fetal brain lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 68 kDa

Observed band size: 68 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-A-Raf antibody [EPR16208] (AB200653)

Anti-A-Raf antibody [EPR16208] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab200653 was shown to bind specifically to A-Raf. A band was observed at 67 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in A-Raf knockout cell line ab286752. To generate this image, wild-type and A-Raf knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-A-Raf antibody [EPR16208] (ab200653) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

A-Raf knockout HCT 116 cell lysate at 20 µg

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

Raji cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 68 kDa

Observed band size: 67 kDa

false

• WB

Lab

Western blot - Anti-A-Raf antibody [EPR16208] (AB200653)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : A-Raf knockout HAP1 cell lysate (20 μg)

Lanes 1 - 2 : Merged signal (red and green). Green - ab200653 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab200653 and a competitor's top cited rabbit polyclonal antibody.

All lanes:

Western blot - Anti-A-Raf antibody [EPR16208] (ab200653)

Predicted band size: 68 kDa

false

• WB

Lab

Western blot - Anti-A-Raf antibody [EPR16208] (AB200653)

Lanes 1-4 : Merged signal (red and green). Green - ab200653 observed at 68 kDa. Red - loading control ab8245 observed at 36 kDa.

ab200653 Anti-A-Raf antibody [EPR16208] was shown to specifically react with A-Raf in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266351 (knockout cell lysate ab257838) was used. Wild-type and A-Raf knockout samples were subjected to SDS-PAGE. ab200653 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-A-Raf antibody [EPR16208] (ab200653) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 2:

A-Raf knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 2:

Western blot - Human A-Raf knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-a-raf-knockout-hek-293t-cell-line-ab266351'>ab266351</a>)

Lane 3:

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

Lane 4:

Raji (Human Burkitts lymphoma cell line) whole 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: 68 kDa

Observed band size: 68 kDa

false

• WB

Lab

Western blot - Anti-A-Raf antibody [EPR16208] (AB200653)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : A-Raf knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : Raji cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab200653 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab200653 was shown to specifically react with A-Raf when A-Raf knockout samples were used. Wild-type and ProteinX knockout samples were subjected to SDS-PAGE. ab200653 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and) Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-A-Raf antibody [EPR16208] (ab200653)

Predicted band size: 68 kDa

false

• WB

Supplier Data

Western blot - Anti-A-Raf antibody [EPR16208] (AB200653)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-A-Raf antibody [EPR16208] (ab200653) at 1/10000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 68 kDa

Observed band size: 68 kDa

false

Exposure time: 1min