Anti-Raf1 antibody [EP4969] - BSA and Azide free

• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - BSA and Azide free (AB236003)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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 (<a href='/en-us/products/primary-antibodies/raf1-antibody-ep4969-n-terminal-ab181115'>ab181115</a>) 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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• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Raf1 antibody [EP4969] - BSA and Azide free (AB236003)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181115).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Raf1 antibody [EP4969] - BSA and Azide free (AB236003)

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labelling with 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181115).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Raf1 antibody [EP4969] - BSA and Azide free (AB236003)

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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181115)

• WB

Lab

Western blot - Anti-Raf1 antibody [EP4969] - BSA and Azide free (AB236003)

This data was developed using the same antibody clone in a different buffer formulation (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 (<a href='/en-us/products/primary-antibodies/raf1-antibody-ep4969-n-terminal-ab181115'>ab181115</a>) 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

Supplier Data

Western blot - Anti-Raf1 antibody [EP4969] - BSA and Azide free (AB236003)

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

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181115).

Lanes 1 - 4:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (<a href='/en-us/products/primary-antibodies/raf1-antibody-ep4969-n-terminal-ab181115'>ab181115</a>) at 1/1000 dilution

Lanes 5 - 6:

Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (<a href='/en-us/products/primary-antibodies/raf1-antibody-ep4969-n-terminal-ab181115'>ab181115</a>)

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

false