Anti-BRAF antibody [EP152Y] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRAF antibody [EP152Y] - BSA and Azide free (AB189351)

This IHC data was generated using the same anti-B Raf antibody clone, EP152Y, in a different buffer formulation (cat# ab33899).

This image shows paraffin embedded human prostate cancer tissue sample stained with ab33899 (unpurified) at 1/250 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRAF antibody [EP152Y] - BSA and Azide free (AB189351)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast cancer tissue sections labeling BRAF with purified ab33899 at 1/100 dilution (4.77 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33899).

• IP

Unknown

Immunoprecipitation - Anti-BRAF antibody [EP152Y] - BSA and Azide free (AB189351)

ab33899 (Purified) at 1 : 500 dilution (0.954 μg/ml) immunoprecipitating BRAF in HeLa whole cell lysate .
Lane 1 (input) : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg
Lane 2 (+) : ab33899 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab33899 in HeLa whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM /TBST .This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189351)

All lanes:

Immunoprecipitation - Anti-BRAF antibody [EP152Y] (<a href='/en-us/products/primary-antibodies/braf-antibody-ep152y-ab33899'>ab33899</a>)

Predicted band size: 84 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRAF antibody [EP152Y] - BSA and Azide free (AB189351)

This IHC data was generated using the same anti-B Raf antibody clone, EP152Y, in a different buffer formulation (cat# ab33899).

ab33899 (unpurified) staining B Raf cells from human prostate tissue by immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). Cells were formaldehyde fixed and permeabilized in PBS-Tween 20 prior to blocking in 70% serum for 10 minutes at 25°C. The primary antibody was diluted 1/250 and incubated with the sample for 1 hour at 25°C. A biotin conjugated goat polyclonal to mouse Ig was used as the secondary.

• WB

Lab

Western blot - Anti-BRAF antibody [EP152Y] - BSA and Azide free (AB189351)

This data was developed using the same antibody clone in a different buffer formulation (ab33899).

Lanes 1- 2 : Merged signal (red and green). Green - ab33899 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab33899 was shown to react with BRAF in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265373 (knockout cell lysate ab257078) was used. Wild-type HeLa and BRAF 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. ab33899 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 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-BRAF antibody [EP152Y] (<a href='/en-us/products/primary-antibodies/braf-antibody-ep152y-ab33899'>ab33899</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

BRAF knockout HeLa cell lysate at 20 µg

Predicted band size: 84 kDa

Observed band size: 90 kDa

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• WB

Lab

Western blot - Anti-BRAF antibody [EP152Y] - BSA and Azide free (AB189351)

Lanes 1 - 3 : Merged signal (red and green). Green - ab33899 (unpurified) observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab33899 was shown to recognize BRAF in wild-type HAP1 cells as signal was lost at the expected MW in BRAF knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and BRAF knockout samples were subjected to SDS-PAGE. ab33899 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 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 (ab33899).

All lanes:

Western blot - Anti-BRAF antibody [EP152Y] (<a href='/en-us/products/primary-antibodies/braf-antibody-ep152y-ab33899'>ab33899</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 40 µg

Lane 2:

BRAF knockout HAP1 whole cell lysate at 40 µg

Lane 3:

HeLa whole cell lysate at 40 µg

Predicted band size: 84 kDa

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