All lanes : Anti-Bcl10 antibody [EP606Y] (ab33905) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BCL10 knockout HeLa cell lysate
Lane 3 : Ramos cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 31 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab33905 observed at 32 kDa. Red - loading control, ab7291 observed at 52 kDa.

ab33905 Anti-Bcl10 antibody [EP606Y] was shown to specifically react with Bcl10 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261797 (knockout cell lysate ab257144) was used. Wild-type and Bcl10 knockout samples were subjected to SDS-PAGE. ab33905 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

 

 

All lanes : Anti-Bcl10 antibody [EP606Y] (ab33905) at 1/5000 dilution (purified)

Lane 1 : Raji cell lysate
Lane 2 : Ramos cell lysate
Lane 3 : HuT-78 cell lysate
Lane 4 : HeLa cell lysate

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: 31 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunofluorescence staining of Raji cells with purified ab33905 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4 % PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab33905 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Immunohistochemical staining of paraffin embedded human lung carcinoma with purified ab33905 at a working dilution of 1/50. The secondary antibody used is ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Flow cytometry analysis of Raji (human Burkitt's lymphoma) cells labeling Bcl10 (red) with ab33905 at a 1/200 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with primary and secondary antibodies.

ab33905 (purified) at 1/20 immunoprecipitating Bcl10 in 10 μg Ramos cell lysate (Lanes 1 and 2, observed at 32 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730). For western blotting, HRP Veriblot for IP (ab131366) was used for detection (1/10 000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

Anti-Bcl10 antibody [EP606Y] (ab33905) at 1/1000 dilution (unpurified) + Recombinant Human Bcl10 protein (ab82241) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 31 kDa


Exposure time: 1 minute

Unpurified ab33905, at a 1/100 dilution, staining human hepatocellular carcinoma by Immunohistochemistry, Paraffin embedded tissue.

Unpurified ab33905, staining HeLa cells by Immunofluorescent.