All lanes : Anti-LMAN1 antibody [EPR6979] (ab125006) at 1/1000 dilution

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Mouse heart lysate
Lane 5 : Rat spleen lysate
Lane 6 : Rat heart lysate
Lane 7 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 58 kDa

Immunocytochemistry analysis of 293T (Human embryonic kidney epithelial cell) cells labeling LMAN1 with Purified ab125006 at 1/50 dilution (2 µg/mL). Cells were fixed in 100% Methanol and permeabilized with None. 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.

All lanes : Anti-LMAN1 antibody [EPR6979] (ab125006) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : LMAN1 knockout HEK-293T cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 58 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab125006 observed at 55 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab125006 Anti-LMAN1 antibody [EPR6979] was shown to specifically react with Protein ERGIC-53 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266248 (knockout cell lysate ab257505) was used. Wild-type and Protein ERGIC-53 knockout samples were subjected to SDS-PAGE. ab125006 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°CC 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 20000 dilution for 1 hour at room temperature before imaging.

 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling LMAN1 with Purified ab125006 at 1/500 dilution (0.21 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.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling LMAN1 with Purified ab125006 at 1/20 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling LMAN1 with Purified ab125006 at 1/500 dilution (0.21 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 LMAN1 with Purified ab125006 at 1/500 dilution (0.21 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.