Antibodies, Proteins, Kits and Reagents for Life Science

Product name

Anti-A RAF antibody [EPR16208]
See all A RAF primary antibodies
Description

Rabbit monoclonal [EPR16208] to A RAF
Host species

Rabbit

Tested Applications & Species

Application	Species
Flow Cyt	Human
ICC/IF	Human
IHC-P	Human
WB	Human

See all applications and species data

Immunogen

Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: HEK-293T, HeLa, Raji, HT-29 and A375 whole cell lysates; Human fetal heart, fetal kidney, fetal brain and bladder lysates. IHC-P: Human cervix carcinoma and kidney tissues. ICC/IF: HeLa and HEK293 cells. Flow Cyt: HeLa cells.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR16208
Isotype

IgG
Research areas

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

All lanes : 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
Lane 2 : ARAF knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : Raji (Human Burkitts lymphoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 68 kDa
Observed band size: 68 kDa

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.
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.
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.
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.
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.
Flow Cytometry - Anti-A RAF antibody [EPR16208] (ab200653)

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

All lanes : Anti-A RAF antibody [EPR16208] (ab200653) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.
Western blot - Anti-A RAF antibody [EPR16208] (ab200653)

All lanes : Anti-A RAF antibody [EPR16208] (ab200653) at 1/1000 dilution

Lane 1 : HT-29 (Human colorectal adenocarcinoma cells) whole cell lysate
Lane 2 : A375 (Human malignant melanoma) whole cell lysate
Lane 3 : Human fetal heart lysate
Lane 4 : Human fetal kidney lysate
Lane 5 : Human bladder lysate

Lysates/proteins at 10 µg per lane.

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

Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.
Western blot - Anti-A RAF antibody [EPR16208] (ab200653)

Anti-A RAF antibody [EPR16208] (ab200653) at 1/1000 dilution + Human fetal brain lysate at 10 µg

Secondary
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

Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.
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.
Anti-A RAF antibody [EPR16208] (ab200653)