Anti-SKP2 antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SKP2 antibody (AB19877)

ab19877 stained in HeLa cells. Cells were fixed with 4% paraformaldehyde (10 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab19877 at 5μg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 μM for 1hour at room temperature.

• WB

Lab

Western blot - Anti-SKP2 antibody (AB19877)

Lanes 1 - 4 : Merged signal (red and green). Green - ab19877 observed at 52 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab19877 was shown to recognize in wild-type HAP1 cells as signal was lost at the expected MW in SKP2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SKP2 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. ab19877 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/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-SKP2 antibody (ab19877) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SKP2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 47 kDa

false

• WB

Unknown

Western blot - Anti-SKP2 antibody (AB19877)

ab19877 detects a band at approximately 46 kDa that corresponds in size to that predicted for SKP2 (48 kDa). This band is detected in several tissue lysates. This antibody was tested in normal liver tissue lysates, but no band was detected.

All lanes:

Western blot - Anti-SKP2 antibody (ab19877) at 1 µg/mL

Lane 1:

Human heart tissue lysate at 20 µg

Lane 2:

Human brain tissue lysate at 20 µg

Lane 3:

Human kidney normal tissue lysate at 20 µg

Lane 4:

Human testis tissue lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

Predicted band size: 47 kDa

Observed band size: 46 kDa

false