All lanes : Anti-Bub1 antibody [EPR18947] (ab195268) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BUB1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 122 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab195268 observed at 125 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab195268 was shown to react with Bub1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265145 (knockout cell lysate ab257373) was used. Wild-type HeLa and BUB1 knockout HeLa cell lysates were subjected to SDS-PAGE. ab195268 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated 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.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Bub1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: F9 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab195268 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab195268 was shown to recognize Bub1 when Bub1 knockout samples were used, along with additional cross-reactive bands. Wild-type and Bub1 knockout samples were subjected to SDS-PAGE. Ab195268 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10,000 dilution respectively and incubated overnight at 4C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Ab195268 staining Bub1 in HeLa (human cervix adenocarcinoma epithelial cell) cells by Immunocytochemistry. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% TritonX-100. Samples were incubated with primary antibody at 1:100 dilution (7.8 μg/ml). Alexa Fluor^® 594 Anti-alpha tubulin [DM1A] – Microtubule Marker (ab195889) was used as the counterstain antibody at 1:200 dilution (2.5 μg/ml).  An AlexaFluor^®488 Goat anti-Rabbit (ab150077) was used as a secondary antibody at 1:1000 dilution (2µg/ml). DAPI was used as a nuclear counterstain. Confocal image showing positive kinetochore staining in HeLa cells in M phase.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Bub1 with ab195268 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on Rat testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Bub1 antibody [EPR18947] (ab195268) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 3 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 122 kDa
Observed band size: 122 kDa

Blocking/Dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: Lane 1 & 2: 10 seconds; Lane 2: 3 minutes.

All lanes : Anti-Bub1 antibody [EPR18947] (ab195268) at 1/1000 dilution

Lane 1 : Human testis lysate
Lane 2 : Human fetal liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 122 kDa
Observed band size: 122 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Bub1 antibody [EPR18947] (ab195268) at 1/1000 dilution

Lane 1 : Mouse testis lysate
Lane 2 : Rat testis lysate
Lane 3 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate
Lane 4 : mESC (Mouse embryonic stem cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 122 kDa
Observed band size: 122 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature PMID:17189386
PMID:16864798

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling Bub1 with ab195268 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on Mouse testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.