All lanes : Anti-Proteasome 20S LMP7 antibody [EPR14482(B)] (ab180606) at 1/1000 dilution

Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : PSMB8 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 4 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 30 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab180606 observed at 23 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab180606 Anti-Proteasome 20S LMP7 antibody [EPR14482(B)] was shown to specifically react with Proteasome 20S LMP7 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267149 (knockout cell lysate ab257130) was used. Wild-type and Proteasome 20S LMP7 knockout samples were subjected to SDS-PAGE. ab180606 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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 gastric carcinoma tissue sections labeling Proteasome 20S LMP7 with purified ab180606 at 1/1700 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed 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.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Proteasome 20S LMP7 with purified ab180606 at 1:100 dilution (8.9 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 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labeling Proteasome 20S LMP7 with purified ab180606 at 1/90 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).

All lanes : Anti-Proteasome 20S LMP7 antibody [EPR14482(B)] (ab180606) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : PSMB8 knockout A549 cell lysate
Lane 3 : Raji cell lysate
Lane 4 : HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 30 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab180606 observed at 23 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab180606 Anti-Proteasome 20S LMP7 antibody [EPR14482(B)] was shown to specifically react with Proteasome 20S LMP7 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267148 (knockout cell lysate ab257129) was used. Wild-type and Proteasome 20S LMP7 knockout samples were subjected to SDS-PAGE. ab180606 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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 20000 dilution for 1 hour at room temperature before imaging.

Anti-Proteasome 20S LMP7 antibody [EPR14482(B)] (ab180606) at 1/10000 dilution (Purified) + Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysates at 15 µg

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

Predicted band size: 30 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded human bladder transitional cell carcinoma tissue labeling Proteasome 20S LMP7 with ab180606 (unpurified) at 1/500 dilution, followed by prediluted ImmunoHistoprobe (Ready to use) HRP Polymer for Rabbit IgG. Counterstained with hematoxylin.

ab180606 (unpurified) staining Proteasome 20S LMP7 in Jurkat (human acute T cell leukemia) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain.

Negative control: PBS only.

Immunofluorecenct analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Proteasome 20S LMP7 with ab180606 (unpurified) at 1/100 dilution, followed by Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody at 1/200 dilution (left panel). DAPI staining (right panel).

Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling Proteasome 20S LMP7 with ab180606 (unpurified) at 1/150 dilution (red) compared to a Rabbit monoclonal IgG Isotype control (green), followed by Goat anti rabbit IgG (FITC) secondary antibody at 1/150 dilution.