Recombinant Anti-A RAF antibody [EPR16208] KO Tested (ab200653)

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

Suitable for: Flow Cyt (Intra), IHC-P, WB, ICC/IFmore details
Species reactivity

Reacts with: Human
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 (intra): 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

Alternative Versions
- Alexa Fluor® 488 Anti-A RAF antibody [EPR16208] (ab214957)
- Anti-A RAF antibody [EPR16208] - BSA and Azide free (ab240354)
Compatible Secondaries
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
KO cell lines
- Human ARAF knockout HEK-293T cell line (ab266351)
KO cell lysates
- Human ARAF knockout HEK-293T cell lysate (ab257838)
Recombinant Protein
- Recombinant human A RAF protein (ab79795)

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab200653 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
Flow Cyt (Intra)		1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IHC-P		1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB		1/1000. Detects a band of approximately 68 kDa (predicted molecular weight: 68 kDa).
ICC/IF		1/250.

Notes
Flow Cyt (Intra) 1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IHC-P 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/1000. Detects a band of approximately 68 kDa (predicted molecular weight: 68 kDa).
ICC/IF 1/250.

Function

Involved in the transduction of mitogenic signals from the cell membrane to the nucleus.
Tissue specificity

Predominantly in urogenital tissues.
Sequence similarities

Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. RAF subfamily.
Contains 1 phorbol-ester/DAG-type zinc finger.
Contains 1 protein kinase domain.
Contains 1 RBD (Ras-binding) domain.
Target information above from: UniProt accession P10398 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 369 Human
- Omim: 311010 Human
- SwissProt: P10398 Human
- Unigene: 446641 Human
Alternative names
- A raf 1 antibody
- A Raf proto oncogene serine/threonine protein kinase antibody
- ARAF 1 antibody
- Araf antibody
- ARaf proto oncogene serine/threonine protein kinase antibody
- ARAF_HUMAN antibody
- ARAF1 antibody
- Oncogene Araf1 antibody
- Oncogene PKS2 antibody
- PKS 2 antibody
- PKS antibody
- PKS2 antibody
- Proto oncogene Pks antibody
- Proto-oncogene A-Raf antibody
- Proto-oncogene A-Raf-1 antibody
- Proto-oncogene Pks antibody
- RAFA 1 antibody
- RAFA1 antibody
- Ras binding protein DA Raf antibody
- Serine/threonine-protein kinase A-Raf antibody
- v raf murine sarcoma 3611 viral oncogene homolog antibody
- v raf murine sarcoma 3611 viral oncogene homolog 1 antibody
- v raf oncogene homolog 1 (murine sarcoma 3611 virus) antibody
see all

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 (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.
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)

Flow cytometry protocols
Immunohistochemistry protocols
Immunocytochemistry & immunofluorescence protocols
Western blot protocols

Click here to view the general protocols

SDS download

Country/Region

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Datasheet download

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Publishing research using ab200653? Please let us know so that we can cite the reference in this datasheet.

ab200653 has been referenced in 4 publications.

Zhang H et al. Knockdown of PLCB2 expression reduces melanoma cell viability and promotes melanoma cell apoptosis by altering Ras/Raf/MAPK signals. Mol Med Rep 21:420-428 (2020). PubMed: 31746389
Yang X et al. Inhibition of ITGB1 enhance the anti-tumor effect of cetuximab in colorectal cancer cell. Medicine (Baltimore) 99:e20944 (2020). PubMed: 32629699
Hao J et al. Antiestrogenic Activity of the Xi-Huang Formula for Breast Cancer by Targeting the Estrogen Receptor a. Cell Physiol Biochem 47:2199-2215 (2018). PubMed: 29975948
Faltermeier CM et al. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis. Proc Natl Acad Sci U S A 113:E172-81 (2016). PubMed: 26621741

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Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Isotype control

KO cell lines

KO cell lysates

Recombinant Protein

Applications

The Abpromise guarantee

Target

Function

Tissue specificity

Sequence similarities

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

Certificate of Compliance

References (4)

Customer reviews and Q&As