Lane 1: Wild-type Hap1 cell lysate, 20 µg

Lane 2: CANX knockout Hap1 cell lysate, 20 µg

Lane 3: Wild-type HEK-293T cell lysate, 20 µg

Lane 4: CANX knockout HEK-293T cell lysate, 20 µg

Lanes 1 - 4: Merged signal (red and green). Green - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) observed at 80 kDa. Red - loading control, ab8245 observed at 37 kDa.  

ab133615 was shown to react with Calnexin in wild-type HEK-293T. Loss of signal was observed when knockout cell line ab255368 (knockout cell lysate ab263805) was used. Wild-type and Calnexin knockout samples were subjected to SDS-PAGE. ab133615 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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.

Overlay histogram showing HeLa cells stained with unpurified ab133615 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab133615, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Lane 1: Wild-type A549 whole cell lysate, 20 μg

Lane 2: CD9 knockout A549 whole cell lysate, 20 μg

Lane 3: MCF7 whole cell lysate, 20 μg

Lanes 1 - 3: Merged signal (red and green). Green - Anti-CD9 antibody [EPR2949] (ab92726) observed at 22 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab92726 was shown to recognize CD9 in wild-type A549 cells as signal was lost at the expected MW in CD9 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CD9 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab92726 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical staining of paraffin embedded rat spleen with purified ab92726 at a working dilution of 1/500. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 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.

Lane 1: Wild-type HAP1 whole cell lysate, 20 µg

Lane 2: Brefeldin A treated wild-type HAP1 whole cell lysate, 20 µg

Lane 3: CD63 knockout HAP1 whole cell lysate, 20 µg

Lane 4: Brefeldin A treated CD63 knockout HAP1 whole cell lysate, 20 µg

Lane 5: HL60 whole cell lysate, 20 µg

Lane 6: Human platelets whole cell lysate, 20 µg

Lanes 1 - 6: Merged signal (red and green). Green - Anti-CD63 antibody [EPR5702] (ab134045) observed at 26 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab134045 was shown to specifically react with CD63 in wild-type HAP1 Brefeldin A treated cells as signal was lost in HAP1 Brefeldin A treated CD63 knockout cells. Wild-type and CD63 knockout samples were subjected to SDS-PAGE. Ab134045 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD63 with purified ab134045 at 1/2000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Primary: Anti-CD81 antibody [EPR4244] (ab109201), unpurified at 1/700 dilution

Lane 1: Ramos cell lysate, 10 µg

Lane 2: U87-MG cell lysate, 10 µg

Lane 3: PC-12 cell lysate, 10 µg

Lane 4: Raw264.7 cell lysate, 10 µg

Secondary: Peroxidase-conjugated goat anti-rabbit IgG (H+L), 1/1000 dilution

Primary: Anti-Hsp70 antibody [EPR16892] (ab181606) at 1/1000 dilution

Lane 1: Human fetal heart lysate, 10 µg

Lane 2: Human fetal kidney lysate, 10 µg

Secondary: Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated, 1/1000 dilution

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Hsp70 with ab181606 at 1/50 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Nuclear and cytoplasm staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (Tubulin mouse mAb) at 1/500 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows;
1. ab181606 at 1/50 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab 150077 (Goat anti rabbit IgG (Alexa Fluor®488) secondary antibody at 1/400 dilution.

Primary: Anti-TSG101 antibody [EPR7130(B)] (ab125011), unpurified at 1/1000 dilution

Lane 1: Human brain lysate, 10 µg

Lane 2: K562 (human chronic myelogenous leukemia cell line from bone marrow) lysate, 10 µg

Lane 3: Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate, 10 µg

Lane 4: SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate, 10 µg

Secondary: HRP-labeled goat anti-rabbit, 1/2000 dilution

Immunocytochemsitry/Immunofluorescence analysis of HEK-293 (human epithelial cell line from embryonic kidney) cells labeling TSG101 (red) with unpurified ab125011 at 1/10. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling TSG101 with unpurified ab125011 at 1/30. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.