Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101)

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling CD9 with ab263019 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on platelets of human spleen. The section was incubated with ab263019 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab263019).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

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.

• WB

Supplier Data

Western blot - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

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.

All lanes:

Western blot - Anti-Hsp70 antibody [EPR16892] (<a href='/en-us/products/primary-antibodies/hsp70-antibody-epr16892-ab181606'>ab181606</a>) at 1/1000 dilution

Lane 1:

Human fetal heart lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 70 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

This data was developed using the same antibody clone in a different buffer formulation (ab263019).

Lanes 1 - 4 : Merged signal (red and green). Green - ab263019 observed at 18 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab263019 was shown to react with CD9 in wild-type HeLa cells in Western blot with loss of signal observed in CD9 knockout cell line ab255375 (CD9 knockout cell lysate ab263754). Wild-type HeLa and CD9 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab263019 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) 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 h at room temperature before imaging.

All lanes:

Western blot - Anti-CD9 antibody [EPR23105-125] (<a href='/en-us/products/primary-antibodies/cd9-antibody-epr23105-125-ab263019'>ab263019</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

CD9 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD9 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cd9-knockout-hela-cell-line-ab255375'>ab255375</a>)

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 25 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

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.

All lanes:

Western blot - Anti-CD63 antibody [EPR5702] - Late Endosome Marker (<a href='/en-us/products/primary-antibodies/cd63-antibody-epr5702-late-endosome-marker-ab134045'>ab134045</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Brefeldin A treated wild-type HAP1 whole cell lysate at 20 µg

Lane 3:

CD63 knockout HAP1 whole cell lysate at 20 µg

Lane 4:

Brefeldin A treated CD63 knockout HAP1 whole cell lysate at 20 µg

Lane 5:

HL60 whole cell lysate at 20 µg

Lane 6:

Human platelets whole cell lysate at 20 µg

Predicted band size: 26 kDa

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• WB

Supplier Data

Western blot - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

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.

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• WB

Supplier Data

Western blot - Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (AB275018)

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

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