Anti-Raf1 antibody [EP4969] - N-terminal

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

Immunofluorescent analysis of acetone-fixed K562 cells labeling Raf1 with unpurified ab181115 at 1/250 dilution, followed by Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody at 1/200 dilution. Counter stained with Dapi.

• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

Western blot : Anti-RAF1 antibody [EP4969] (ab181115) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab181115 was shown to bind specifically to RAF1. A band was observed at 73 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in RAF1 knockout cell line. To generate this image, wild-type and RAF1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human RAF1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-raf1-knockout-hct116-cell-line-ab286619'>ab286619</a>)

Lane 2:

RAF1 knockout HCT 116 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 73 kDa

Observed band size: 73 kDa

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• WB

Supplier Data

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

All lanes:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 20 µg

Lane 2:

K562 cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 73 kDa

Observed band size: 73 kDa

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• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

All lanes:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 73 kDa

Observed band size: 73 kDa

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• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

ab181602 was used as GAPDH loading control. ab181115 was used to detect Raf1. ab150365 was used to detect Raf1 phospho S43.

All lanes:

Purified at 1/5000 dilution

Lane 1:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 15 µg

Lane 2:

HEK-293 (Human embryonic kidney epithelial cell) treated with 100nM Phorbol-12-myristate-13-acetate for 1 hour whole cell lysates at 15 µg

Lane 3:

HEK-293 (Human embryonic kidney epithelial cell) treated with 100nM Phorbol-12-myristate-13-acetate for 1 hour whole cell lysates, then incubated with phosphatase at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 73 kDa

Observed band size: 73 kDa

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• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

All lanes:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution

All lanes:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 73 kDa

Observed band size: 73 kDa

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• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labelling with unpurified ab181115 at 1/100 dilution (10.79μg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Raf1 with purified ab181115 at 1/100 dilution (10 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• WB

Supplier Data

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

Lanes 1 and 2 : Green signal from target – ab181115 observed at 74 kDa
Lanes 3 and 4 : Red signal from loading control – ab8226 observed at 42 kDa
Lanes 5 and 6 : Merged (red and green) signal

Unpurified ab181115 was shown to specifically react with Raf1 when Raf1 knockout samples were used. Wild-type and Raf1 knockout samples were subjected to SDS-PAGE. ab181115 and ab8226 (loading control to beta actin) were both diluted 1/1000 and 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^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 4:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution

Lanes 5 - 6:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115)

Lanes 1, 3 and 5:

Wild-type HAP1 cell lysate at 20 µg

Lanes 2, 4 and 6:

Raf1 knockout HAP1 cell lysate at 20 µg

Predicted band size: 73 kDa

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• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

Lanes 1- 2 : Merged signal (red and green). Green - ab181115 observed at 73 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab181115 was shown to react with Raf1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264978 (knockout cell lysate ab257126) was used. Wild-type HeLa and Raf1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab181115 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 20000 dilution and a 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.

All lanes:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/20000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RAF1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human RAF1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-raf1-knockout-hela-cell-line-ab264978'>ab264978</a>)

Predicted band size: 73 kDa

Observed band size: 73 kDa

false

• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

All lanes:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution

Lane 1:

RAW 264.7(Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Lane 2:

C2C12 (Mouse myoblasts myoblast) whole cell lysates at 20 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 73 kDa

Observed band size: 73 kDa

false

• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

Lanes 1 : Wild-type HAP1 cell lysate (20 μg)
Lanes 2 : Raf1 knockout HAP1 cell lysate (20 μg)
Lanes 1 and 2 : Merged signal (red and green).

Green - Target observed at 74 kDa. Red - loading control, ab8226, observed at 42 kDa

This western blot image is a comparison between unpurified ab181115 and a competitor's top cited discontinued rabbit polyclonal antibody.

All lanes:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115)

Predicted band size: 73 kDa

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