Raf-1 Antibody (563002)

R&D Systems | Catalog # MAB4540

R&D Systems

Key Product Details

Species Reactivity

Validated:

Human, Mouse, Rat

Cited:

Human, Mouse

Applications

Validated:

Immunohistochemistry, Western Blot, Simple Western

Cited:

Western Blot

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG2A Clone # 563002
View all Raf-1 Primary Antibodies »

Product Specifications

Immunogen

E. coli-derived recombinant human Raf-1
Asn189-Thr353
Accession # P04049

Specificity

Detects human, mouse, and rat Raf-1 at 74 kDa in Western blots.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG2A

Scientific Data Images for Raf-1 Antibody (563002)

Detection of Human, Mouse, and Rat Raf‑1 antibody by Western Blot.

Detection of Human, Mouse, and Rat Raf‑1 by Western Blot.

Western blot shows lysates of MCF-7 human breast cancer cell line, HepG2 human hepatocellular carcinoma cell line, A431 human epithelial carcinoma cell line, L-929 mouse fibroblast cell line, and NRK rat normal kidney cell line. PVDF Membrane was probed with 1 µg/mL of Mouse Anti-Human/Mouse/Rat Raf-1 Monoclonal Antibody (Catalog # MAB4540) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for Raf-1 at approximately 74 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 3.
Raf-1 antibody in Human Liver by Immunohistochemistry (IHC-P).

Raf‑1 in Human Liver.

Raf-1 was detected in immersion fixed paraffin-embedded sections of human liver array using Mouse Anti-Human/Mouse/Rat Raf-1 Monoclonal Antibody (Catalog # MAB4540) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS002) and counterstained with hematoxylin (blue). Lower panel shows a lack of labeling if primary antibodies are omitted and tissue is stained only with secondary antibody followed by incubation with detection reagents. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human Raf‑1 by Simple WesternTM.

Simple Western lane view shows lysates of MCF‑7 human breast cancer cell line, loaded at 0.2 mg/mL. A specific band was detected for Raf‑1 at approximately 75 kDa (as indicated) using 10 µg/mL of Mouse Anti-Human/Mouse/Rat Raf‑1 Monoclonal Antibody (Catalog # MAB4540). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.
Detection of Raf-1 by Western Blot

Detection of Raf-1 by Western Blot

CRAF activity is sufficient to drive macropinocytosis. Overexpression of constitutively active mutants of the CRAF kinase (S259A or S257L) activates the EV uptake, causes phenotypic transformation and triggers formation of ruffle structures. (a) Western blotting documents overexpression of the CRAF kinase mutants in IEC18 cells including IEC18‐CRAF/S259A and IEC18‐CRAF/S257L cells. (b) Flow cytometry indicates the increased uptake of fluorescent RAS3 EVs by IEC18‐CRAF/S259A and IEC18‐CRAF/S257L relative to RAS3 cells. CRAF mediated EV uptake is obliterated by a specific CRAF inhibitor GW5407 (flow cytometry, three biological replicates for RAS3 and GW5074‐treated cells and six for control CRAF mutant IEC18 cells). (c) Confocal images illustrating a dramatic morphological transformation coupled with the generation of ruffle structures and increased uptake of fluorescent EVs by IEC18‐CRAF/S259A and IEC18‐CRAF/S257L cell lines compared to parental IEC18 cells. (d) Confocal images of immunofluorescent staining show that intracellular distribution of the endogenous RAS protein is re‐organized following activated CRAF expression. RAS signal was enhanced and clustered around ruffle region in contrast to its dimmer appearance and distribution around plasma membrane in non‐transformed cells Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34136107), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Raf-1 by Western Blot

Detection of Raf-1 by Western Blot

CRAF is required for particle macropinocytosis by HRAS transformed cells. (a) Western blotting documenting the downregulation of CRAF by siRNA in RAS3 cells. (b) Flow cytometry illustrates the reduced EV uptake by RAS3 cells following CRAF down‐regulation. (c to e) Confocal images documenting the decreased 70‐kDa dextran and EV uptake in CRAF siRNA‐treated RAS3 cells (c). However, transferrin uptake was not affected in CRAF siRNA treated RAS3 cells (d). Quantification of EV uptake in confocal images; fluorescent deposit counting was conducted in 3 randomly taken images of individual cells Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34136107), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Raf-1 by Immunocytochemistry/ Immunofluorescence

Detection of Raf-1 by Immunocytochemistry/ Immunofluorescence

Signalling intermediates of the RAS/CRAF macropinocytosis pathway and its impact on experimental metastasis. (a) Phosphorylation of myosin phosphatase (MYPT) in cells harbouring transformed CRAF and RAS. Confocal images point to co‐localization between CRAF and pMYPT (T696) in IEC18‐CRAF/S257L and RAS3 cells. Additional co‐localization images were represented in Figure S9. (b and c) Metastasis assays: Control and EIPA pre‐treated (19 h) RAS3 cells were injected to the YFP‐SCID mouse via tail vein (n = 8 for control and n = 9 for EIPA pre‐treated RAS3 cells). After 4 weeks, mice were sacrificed and lung tissues were extracted to enumerate metastatic nodules. Additional biological replicate in NSG mice (n = 7) are presented in Figure S12C Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34136107), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Raf-1 by Immunocytochemistry/ Immunofluorescence

Detection of Raf-1 by Immunocytochemistry/ Immunofluorescence

CRAF activity is sufficient to drive macropinocytosis. Overexpression of constitutively active mutants of the CRAF kinase (S259A or S257L) activates the EV uptake, causes phenotypic transformation and triggers formation of ruffle structures. (a) Western blotting documents overexpression of the CRAF kinase mutants in IEC18 cells including IEC18‐CRAF/S259A and IEC18‐CRAF/S257L cells. (b) Flow cytometry indicates the increased uptake of fluorescent RAS3 EVs by IEC18‐CRAF/S259A and IEC18‐CRAF/S257L relative to RAS3 cells. CRAF mediated EV uptake is obliterated by a specific CRAF inhibitor GW5407 (flow cytometry, three biological replicates for RAS3 and GW5074‐treated cells and six for control CRAF mutant IEC18 cells). (c) Confocal images illustrating a dramatic morphological transformation coupled with the generation of ruffle structures and increased uptake of fluorescent EVs by IEC18‐CRAF/S259A and IEC18‐CRAF/S257L cell lines compared to parental IEC18 cells. (d) Confocal images of immunofluorescent staining show that intracellular distribution of the endogenous RAS protein is re‐organized following activated CRAF expression. RAS signal was enhanced and clustered around ruffle region in contrast to its dimmer appearance and distribution around plasma membrane in non‐transformed cells Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34136107), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Raf-1 Antibody (563002)

Application
Recommended Usage

Immunohistochemistry

8-25 µg/mL
Sample: Immersion-fixed paraffin-embedded sections of human liver and human liver cancer tissue

Simple Western

10 µg/mL
Sample: MCF‑7 human breast cancer cell line

Western Blot

1 µg/mL
Sample: MCF‑7 human breast cancer cell line, HepG2 human hepatocellular carcinoma cell line, A431 human epithelial carcinoma cell line, L‑929 mouse fibroblast cell line, and NRK rat normal kidney cell line

Reviewed Applications

Read 3 reviews rated 4.3 using MAB4540 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein A or G purified from hybridoma culture supernatant

Reconstitution

Reconstitute at 0.5 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Raf-1

The Raf serine/threonine kinases are effectors of Ras that function as MAP3Ks in the ERK phosphorylation cascade. Mammals express three Raf proteins: A-Raf, B-Raf, and Raf-1, also known as C-Raf. Human Raf-1 contains three distinct regions; an N-terminal RBD (Ras-binding domain) (aa 56 - 131), followed by two rich segments [a cysteine-finger region (aa 138 - 184) (also called CR1/C1) and a second cysteine-rich region (CR2) (aa 253 - 264)] and a C-terminal Ser/Thr kinase catalytic domain (aa 354 - 611). Active Raf-1 phosphorylates and activates the MAPK kinases MEK1 and 2, which in turn phosphorylate and activate the MAP kinases ERK1 and 2.

Long Name

v-raf-1Murine Leukemia Viral Oncogene Homolog 1

Alternate Names

c-Raf-1, Raf1, v-Raf-1

Entrez Gene IDs

5894 (Human); 110157 (Mouse); 24703 (Rat)

Gene Symbol

RAF1

UniProt

P04049

Additional Raf-1 Products

Product Documents for Raf-1 Antibody (563002)

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Product Specific Notices for Raf-1 Antibody (563002)

For research use only

Citations for Raf-1 Antibody (563002)

Customer Reviews for Raf-1 Antibody (563002) (3)

4.3 out of 5
3 Customer Ratings
5 Stars
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4 Stars
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3 Stars
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Customer Images

Showing  1 - 3 of 3 reviews Showing All
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  • Human/Mouse/Rat Raf-1 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: Lung cells
    Species: Mouse
    Verified Customer | Posted 10/26/2021
    Raf-1 Antibody (563002) MAB4540
  • Human/Mouse/Rat Raf-1 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: Hematological cancer cell lines lysates
    Species: Human
    Verified Customer | Posted 11/18/2019
    30 ug of protein per sample. 1:500 dilution of primary Ab.
    Raf-1 Antibody (563002) MAB4540
  • Human/Mouse/Rat Raf-1 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: Lung single-cell suspension
    Species: Mouse
    Verified Customer | Posted 06/06/2019
    Raf-1 Antibody (563002) MAB4540

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