Anti-BRAF antibody [EP152Y]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRAF antibody [EP152Y] (AB33899)

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 heat mediated antigen retrieval 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRAF antibody [EP152Y] (AB33899)

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

• IP

Unknown

Immunoprecipitation - Anti-BRAF antibody [EP152Y] (AB33899)

ab33899 (Purified) at 1/20 dilution (20 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, ab33899 at 1/500 and VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1/1000 dilution.
Blocking and diluting buffer : 5% NFDM /TBST .

All lanes:

Immunoprecipitation - Anti-BRAF antibody [EP152Y] (ab33899)

Predicted band size: 84 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRAF antibody [EP152Y] (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] (AB33899)

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 B Raf in wild-type HAP1 cells as signal was lost at the expected MW in B Raf knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and B Raf 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.

All lanes:

Western blot - Anti-BRAF antibody [EP152Y] (ab33899) 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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• WB

Lab

Western blot - Anti-BRAF antibody [EP152Y] (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] (ab33899) 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

Supplier Data

Western blot - Anti-BRAF antibody [EP152Y] (AB33899)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The bands below 90 kDa in lane 1 may be caused by degradation. Lysates were freshly made and used immediately to minimize protein degradation. In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Anti-BRAF antibody (ab33899) total protein control staining at 1/1000 dilution. Exposure time : 3 minutes

All lanes:

Western blot - Anti-BRAF (phospho S446) antibody [EPR28069-63] (<a href='/en-us/products/primary-antibodies/braf-phospho-s446-antibody-epr28069-63-ab313793'>ab313793</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

BRAF knockout HAP1 whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Wild-type HAP1 whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 4:

BRAF knockout HAP1 whole cell lysate (phosphatase treated membrane) 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 1/100000 dilution

Observed band size: 90 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-BRAF antibody [EP152Y] (AB33899)

All lanes:

Western blot - Anti-BRAF antibody [EP152Y] (ab33899) at 1/2000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

Mouse brain lysates 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: 84 kDa

Observed band size: 87 kDa

false

• WB

Unknown

Western blot - Anti-BRAF antibody [EP152Y] (AB33899)

All lanes:

Western blot - Anti-BRAF antibody [EP152Y] (ab33899) at 1/1000 dilution

Lane 1:

Lysate prepared from mouse brain

Lane 2:

Lysate prepared from mouse heart

Lane 3:

Lysate prepared from mouse kidney

Lane 4:

Lysate prepared from mouse spleen

Lane 5:

Lysate prepared from rat brain

Lane 6:

Lysate prepared from rat heart

Lane 7:

Lysate prepared from rat kidney

Lane 8:

Lysate prepared from rat spleen

Predicted band size: 84 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-BRAF antibody [EP152Y] (AB33899)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The band below 90 kDa in lane 1 may be caused by degradation. Lysates were freshly made and used immediately to minimize protein degradation. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Anti-BRAF antibody (ab33899) total protein control staining at 1/1000 dilution. Exposure time : Lane 1, 2 : 3 minutes; Lane 3, 4 : 15 seconds

All lanes:

Western blot - Anti-BRAF (phospho S446) antibody [EPR28069-63] (<a href='/en-us/products/primary-antibodies/braf-phospho-s446-antibody-epr28069-63-ab313793'>ab313793</a>) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

NIH/3T3 whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate (untreated membrane) at 20 µg

Lane 4:

PC-12 whole cell lysate (phosphatase treated membrane) 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 1/100000 dilution

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-BRAF antibody [EP152Y] (AB33899)

All lanes:

Western blot - Anti-BRAF antibody [EP152Y] (ab33899) at 1/5000 dilution

All lanes:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates 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: 84 kDa

Observed band size: 87 kDa

false

• WB

Unknown

Western blot - Anti-BRAF antibody [EP152Y] (AB33899)

All lanes:

Western blot - Anti-BRAF antibody [EP152Y] (ab33899) at 1/5000 dilution

All lanes:

HeLa cell lysate

Predicted band size: 84 kDa

Observed band size: 87 kDa

false

• WB

CiteAb

Western blot - Anti-BRAF antibody [EP152Y] (AB33899)

BRAF western blot using anti-BRAF antibody [EP152Y] ab33899. Publication image and figure legend from Zhang, H., Zheng, D., et al., 2016, Sci Rep, PubMed 27767059.

ab33899 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab33899 please see the product overview.

Compounds 1, 2, and 9 downregulate RAF-MAPK signaling pathway and snail protein level.RAS-RAF-MEK-ERK cascades and snail protein were analyzed by western blot. TE1 cells were treated with 1, 2, or 9 with various concentrations (0, 10, 20 μM) for 48 h. Sorafenib (SFB) was used as the positive control.

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