- 44-502 - Invitrogen Antibodies RAF1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Phospho-c-Raf (Ser259) was done on 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were subsequently labeled with Phospho-c-Raf (Ser259) Polyclonal Antibody (Product # 44-502) at 1:100 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature. Nuclei (Blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Phospho-c-Raf (Ser259) signal was observed in EGF treated control and scrambled cell lines (panels a and b) and not in the EGFR knockout (KO) cell line (panel c). Panels d-f represent the respective untreated cells. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Phospho-c-Raf (Ser259) was performed using 70% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-c-Raf (Ser259) Polyclonal Antibody (Product # 44-502) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree Celsius and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membrane localization. Panel e shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of c-Raf [pS259] was done on A549 cells treated with EGF (200ng/mL, 10 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with c-Raf [pS259] Rabbit Polyclonal Antibody (44502, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Total cell lysates (1) or immunoprecipitates (2-7) prepared from Hek293 cells overexpressing c-Raf and stimulated with EGF were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either untreated (1-6), or treated with lambda (λ) phosphatase (7), blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with 1:1000 dilution of L c-Raf (pS259) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2, 6, 7), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F (ab’)2 anti-rabbit IgG alkaline phosphatase (Product # ALI4405), and signals were detected using the Tropix WesternStar™ method. The data show that only the peptide corresponding to c-Raf (pS 259) blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

44-502