Datasheet

Biological Description

Specificity	c-Raf Antibody [K20J19] detects endogenous levels of total c-Raf protein.
Background	c-Raf (Raf-1), together with A-Raf and B-Raf, forms the Raf kinase family of serine/threonine kinases within the TKL group, serving as major effectors recruited by GTP-bound Ras to initiate the MEK-ERK MAPK pathway that orchestrates key cellular processes including proliferation, survival, differentiation, and migration. c-Raf features an N-terminal regulatory region with a Ras-binding domain (RBD) and a cysteine-rich C1 domain for Ras-dependent membrane localization, a hinge region, and a C-terminal kinase domain containing pivotal activation loop phosphorylation sites (Thr491, Ser494, Ser497, Ser499) as well as priming sites Ser338 (phosphorylated by PAK) and Tyr341 (by Src family kinases); its activity is further modulated by inhibitory 14-3-3 binding motifs at Ser259 (Akt-regulated) and Ser621 (AMPK-regulated). Activation of c-Raf is driven by multi-site phosphorylation, which induces conformational changes that facilitate high-affinity MEK1/2 binding and phosphorylation, thereby amplifying ERK1/2 signaling to drive cell cycle progression (via cyclin D), inhibit apoptosis (through Bad phosphorylation), promote cytoskeletal remodeling, and modulate transcription factors such as Elk-1. c-Raf integrates growth factor and Ras inputs into dynamic pathway flux, but sustained MEK-ERK signaling triggers feedback hyperphosphorylation at multiple inhibitory sites (Ser29, Ser43, Ser289, Ser296, Ser301, Ser642), dampening its responsiveness. Dysregulated c-Raf contributes to oncogenesis in Ras-mutant tumors (such as pancreatic and lung cancers), can be paradoxically activated by some inhibitors, and, in contrast to B-Raf^V600E’s constitutive activity in melanoma, requires strict Ras/Src-dependent activation and exhibits lower basal activity.

Specificity

c-Raf Antibody [K20J19] detects endogenous levels of total c-Raf protein.

Background

c-Raf (Raf-1), together with A-Raf and B-Raf, forms the Raf kinase family of serine/threonine kinases within the TKL group, serving as major effectors recruited by GTP-bound Ras to initiate the MEK-ERK MAPK pathway that orchestrates key cellular processes including proliferation, survival, differentiation, and migration. c-Raf features an N-terminal regulatory region with a Ras-binding domain (RBD) and a cysteine-rich C1 domain for Ras-dependent membrane localization, a hinge region, and a C-terminal kinase domain containing pivotal activation loop phosphorylation sites (Thr491, Ser494, Ser497, Ser499) as well as priming sites Ser338 (phosphorylated by PAK) and Tyr341 (by Src family kinases); its activity is further modulated by inhibitory 14-3-3 binding motifs at Ser259 (Akt-regulated) and Ser621 (AMPK-regulated). Activation of c-Raf is driven by multi-site phosphorylation, which induces conformational changes that facilitate high-affinity MEK1/2 binding and phosphorylation, thereby amplifying ERK1/2 signaling to drive cell cycle progression (via cyclin D), inhibit apoptosis (through Bad phosphorylation), promote cytoskeletal remodeling, and modulate transcription factors such as Elk-1. c-Raf integrates growth factor and Ras inputs into dynamic pathway flux, but sustained MEK-ERK signaling triggers feedback hyperphosphorylation at multiple inhibitory sites (Ser29, Ser43, Ser289, Ser296, Ser301, Ser642), dampening its responsiveness. Dysregulated c-Raf contributes to oncogenesis in Ras-mutant tumors (such as pancreatic and lung cancers), can be paradoxically activated by some inhibitors, and, in contrast to B-Raf^V600E’s constitutive activity in melanoma, requires strict Ras/Src-dependent activation and exhibits lower basal activity.

Usage Information

Application

WB

Dilution

WB

1:1000

Reactivity

Human, Mouse, Rat, Monkey, Bovine, Pig

Source

Mouse Monoclonal Antibody

MW

75 kDa

Storage Buffer

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage
(from the date of receipt)

-20°C (avoid freeze-thaw cycles), 2 years

Exprimental Methods
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Exprimental Methods

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

References

https://pubmed.ncbi.nlm.nih.gov/28694330/
https://pubmed.ncbi.nlm.nih.gov/29358316/

c-Raf Antibody [K20J19]

Biological Description

Usage Information

References

Application Data