All lanes : Anti-BRAF antibody [EP152Y] (ab33899) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 85 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

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.

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.

All lanes : Anti-BRAF antibody [EP152Y] (ab33899) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : B Raf knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 85 kDa

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.

Anti-BRAF antibody [EP152Y] (ab33899) at 1/5000 dilution (Purified) + PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 15 µg

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

Predicted band size: 85 kDa
Observed band size: 87 kDa why is the actual band size different from the predicted?

All lanes : Anti-BRAF antibody [EP152Y] (ab33899) at 1/2000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Mouse brain lysates

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 85 kDa
Observed band size: 87 kDa why is the actual band size different from the predicted?

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 .

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

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.

See Abreview

Anti-BRAF antibody [EP152Y] (ab33899) at 1/5000 dilution (unpurified) + HeLa cell lysate

Predicted band size: 85 kDa
Observed band size: 87 kDa why is the actual band size different from the predicted?

All lanes : Anti-BRAF antibody [EP152Y] (ab33899) at 1/1000 dilution (unpurified)

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: 85 kDa


Exposure time: 3 minutes