Anti-CDC42 antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CDC42 antibody (AB64533)

ICC/IF image of ab64533 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab64533, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive result in 4% pfa fixed (10 min) HeLa cells at 5µg/ml.

• WB

Lab

Western blot - Anti-CDC42 antibody (AB64533)

Lanes 1 - 4 : Merged signal (red and green). Green – ab64533 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab64533 was shown to recognize CDC42 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when CDC42 knockout samples were eexamined. Wild-type and CDC42 knockout samples were subjected to SDS-PAGE. ab64533 and ab8245 (loading control to GAPDH) were diluted at 1μg/ml and 1/10,000 respectively and incubated overnight at 4°C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CDC42 antibody (ab64533) at 1 µg/mL

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

CDC42 knockout HAP1 cell lysate at 20 µg

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Predicted band size: 21 kDa

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• WB

Unknown

Western blot - Anti-CDC42 antibody (AB64533)

Lanes 1-4 : Merged signal (red and green). Green - ab64533 observed at 20 kDa. Red - loading control ab8245 observed at 37 kDa.

ab64533 Anti-CDC42 antibody was shown to specifically react with CDC42 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266522 (knockout cell lysate ab256868) was used. Wild-type and CDC42 knockout samples were subjected to SDS-PAGE. ab64533 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 μg/ml 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.

All lanes:

Western blot - Anti-CDC42 antibody (ab64533) at 1 µg/mL

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

CDC42 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human CDC42 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-cdc42-knockout-hek-293t-cell-line-ab266522'>ab266522</a>)

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 20 kDa

false

• WB

Unknown

Western blot - Anti-CDC42 antibody (AB64533)

All lanes:

Western blot - Anti-CDC42 antibody (ab64533) at 1 µg/mL

Lane 1:

Western blot - Recombinant Human CDC42 protein (T7 tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-cdc42-protein-ab87713'>ab87713</a>) at 0.1 µg

Lane 2:

Western blot - Recombinant Human CDC42 protein (T7 tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-cdc42-protein-ab87713'>ab87713</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 21 kDa

true

Exposure time: 1min

• WB

Unknown

Western blot - Anti-CDC42 antibody (AB64533)

All lanes:

Western blot - Anti-CDC42 antibody (ab64533) at 1 µg/mL

Lane 1:

Western blot - Recombinant Human CDC42 protein (T7 tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-cdc42-protein-ab87713'>ab87713</a>) at 0.1 µg

Lane 2:

Western blot - Recombinant Human CDC42 protein (T7 tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-cdc42-protein-ab87713'>ab87713</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 21 kDa

true

Exposure time: 1min

• WB

Project

Western blot - Anti-CDC42 antibody (AB64533)

All lanes:

Western blot - Anti-CDC42 antibody (ab64533) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

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

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 5:

U2OS (Human osteosarcoma 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: 21 kDa

Observed band size: 21 kDa,52 kDa

true

Exposure time: 3min