All lanes : Anti-DIAPH1 antibody [EPR7948] (ab129167) at 1/10000 dilution (Purified)

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma ) whole cell lysate
Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 141 kDa

All lanes : Anti-DIAPH1 antibody [EPR7948] (ab129167) at 1/1000 dilution (Purified)

Lane 1 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : Human brain lysate
Lane 3 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 141 kDa

All lanes : Anti-DIAPH1 antibody [EPR7948] (ab129167) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : DIAPH1 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 141 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab129167 observed at 150 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab129167 was shown to react with DIAPH1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266120 (knockout cell lysate ab257411) was used. Wild-type HEK-293T and DIAPH1 knockout HEK-293T 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. ab129167 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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.

Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling DIAPH1 with Purified ab129167 at 1/50 dilution (10 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 dilution (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/mL). DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling DIAPH1 with Purified ab129167 at 1/100 dilution (5.96 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling DIAPH1 with Purified ab129167 at 1/100 dilution (5.96 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling DIAPH1 with Purified ab129167 at 1/100 dilution (5.96 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-DIAPH1 antibody [EPR7948] (ab129167) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : DIAPH1 knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 141 kDa
Observed band size: 155 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab129167 observed at 155 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab129167 was shown to react with DIAPH1 in HCT 116 wild-type cells in western blot with loss of signal observed in DIAPH1 knockout cell line ab273727 (DIAPH1 knockout cell lysate ab275252). HCT 116 wild-type and DIAPH1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab129167 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

Flow Cytometry analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling DIAPH1 with purified ab129167 at 1/50 dilution. Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 was used as the secondary antibody (red). Rabbit monoclonal IgG (ab172730) was used as the isotype control (black). Cells without incubation with primary and secondary antibodies were used as the unlabeled control (blue).