All lanes : Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/20000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : RAF1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 73 kDa
Observed band size: 73 kDa

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.

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.

Lanes 1-2 : Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution
Lanes 3-4 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/1000 dilution


Lanes 1 & 3 & 5 : Wild-type HAP1 cell lysate
Lanes 2 & 4 & 6 : Raf1 knockout HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 73 kDa

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 4ºC. 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: 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.

Flow cytometry analysis of K-562 ( Human chronic myelogenous leukemia lymphoblast) cells labelling with unpurified ab181115 at 1/100 dilution (10.79 µg/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.

Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution (Purified) + PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 73 kDa
Observed band size: 73 kDa

All lanes : Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution (Purified)

Lane 1 : RAW 264.7(Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 2 : C2C12 (Mouse myoblasts myoblast) whole cell lysates
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 73 kDa
Observed band size: 73 kDa

All lanes : Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 73 kDa
Observed band size: 73 kDa

All lanes : Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution ((unpurified))

Lane 1 : HeLa cell lysate
Lane 2 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

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

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.