- PA5-29333 - Invitrogen Antibodies RAF1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of RAF1 using 30 µg of A431 lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a RAF1 polyclonal antibody (Product # PA5-29333) at a dilution of 1:5000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of c-Raf was performed by separating 30 µg of various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a c-Raf Polyclonal Antibody (Product # PA5-29333) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of c-Raf was achieved by transfecting HeLa cells with c-Raf specific validated siRNAs (Silencer® select Product # s11750). Western blot analysis (Fig a) was performed using membrane extract from the RAF1 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-c-Raf Rabbit Polyclonal Antibody (Product # PA5-29333, 1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to c-Raf.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), MDA-MB-231 (Lane 3), T-47D (Lane 4) and Ramos (Lane 5). The blot was probed with Rabbit Anti-c-Raf Polyclonal Antibody (Product # PA5-29333, 1:2500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Two isoforms at 72 and 75 kDa corresponding to c-Raf were observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of c-Raf was performed by separating 30 µg of non-transfected (–) and transfected (+) 293T whole cell extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a c-Raf Polyclonal Antibody (Product # PA5-29333) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of RAF1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR627776_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of RAF1 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa RAF1 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with c-Raf Polyclonal Antibody (Product # PA5-29333, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 (Product # A44115). Chemiluminescent detection was performed usingSuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to RAF1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of c-Raf was performed by separating 30 µg of various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a c-Raf Polyclonal Antibody (Product # PA5-29333) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of RAF1 in methanol-fixed HeLa cells using a RAF1 polyclonal antibody (Product # PA5-29333) at a 1:200 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded MCF7 xenograft, using Raf1 (Product # PA5-29333) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-29333