Anti-CD63 antibody

• WB

Lab

Western blot - Anti-CD63 antibody (AB68418)

Lanes 1 - 6 : Merged signal (red and green). Green - ab68418 observed at 26 kDa. Red - loading control, ab8245, observed at 38 kDa.

ab68418 was shown to recognize CD63 in wild-type HAP1 as signal was lost at the expected MW in CD63 knockout HAP1 cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CD63 knockout samples were subjected to SDS-PAGE. ab68418 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml 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 (ab68418) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Wild-type HAP1 Breferldin A treated whole cell lysate at 20 µg

Lane 3:

CD63 knockout HAP1 whole cell lysate at 20 µg

Lane 4:

CD63 knockout HAP1 Breferldin A treated 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

false

• WB

Ap

Western blot - Anti-CD63 antibody (AB68418)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab68418 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

CD63 has a predicted molecular weight of 25 kDa; however it has a number of potential glycosylation sites which may affect the migration of the protein (SwissProt data). This is consistent with the literature which describes a band between 30-60 kDa in human platelets(PMID : 1993697).

All lanes:

Western blot - Anti-CD63 antibody (ab68418) at 1 µg/mL

All lanes:

Platelet (Human) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 26 kDa

Observed band size: 43 kDa

true

Exposure time: 8min

• WB

Unknown

Western blot - Anti-CD63 antibody (AB68418)

The 42 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to human CD63 antigen.

All lanes:

Western blot - Anti-CD63 antibody (ab68418) at 1 µg/mL

All lanes:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 26 kDa

Observed band size: 145 kDa,24 kDa,42 kDa

true

Exposure time: 20min