Anti-CDK5 antibody [EP715Y]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CDK5 antibody [EP715Y] (AB40773)

ab40773 was shown to react with CDK5 in wild-type HEK293T cells in immunocytochemistry with loss of signal observed in CDK5 knockout cell line ab266089. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab40773 at 1/250 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor^® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CDK5 antibody [EP715Y] (AB40773)

Immunocytochemistry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling CDK5 with Purified ab40773 at 1/50 dilution (4.6 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CDK5 antibody [EP715Y] (AB40773)

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling CDK5 with Purified ab40773 at 1 : 20 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor™ 488, ab150077) secondary antibody was used at 1 : 2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IP

Lab

Immunoprecipitation - Anti-CDK5 antibody [EP715Y] (AB40773)

Purified ab40773 at 1 : 20 dilution (1μg) immunoprecipitating CDK5 in SH-SY5Y whole cell lysate.
Lane 1 (input) : SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate 10μg.
Lane 2 (+) : ab40773 + SH-SY5Y whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab40773 in SH-SY5Y whole cell lysate.
VeriBlot for IP Detection Reagent (HRP)™(ab131366) (1 : 1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 33 kDa

All lanes:

Immunoprecipitation - Anti-CDK5 antibody [EP715Y] (ab40773)

Predicted band size: 33 kDa

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• WB

Unknown

Western blot - Anti-CDK5 antibody [EP715Y] (AB40773)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CDK5 antibody [EP715Y] (ab40773) at 1/2000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma ) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

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• WB

Supplier Data

Western blot - Anti-CDK5 antibody [EP715Y] (AB40773)

ab40773 was shown to react with CDK5 in wild-type HEK293T cells in Western blot with loss of signal observed in a CDK5 knockout cell line. Wild-type HEK293T and CDK5 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab40773 overnight at 4 °C at a 1/2000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-CDK5 antibody [EP715Y] (ab40773) at 1/2000 dilution

Lane 1:

Wild-type HAP1 lysate at 30 µg

Lane 2:

CDK5 knock-out HAP1 lysate at 30 µg

Lane 3:

Wild-type HEK293T lysate at 30 µg

Lane 4:

CDK5 knock-out HEK293T lysate at 30 µg

Observed band size: 33 kDa

false

• WB

Lab

Western blot - Anti-CDK5 antibody [EP715Y] (AB40773)

Lanes 1 - 4 : Merged signal (red and green). Green - ab40773 observed at 33 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab40773 was shown to specifically react with Cdk5 in wild-type HAP1 cells as signal was lost in CDK5 knockout cells. Wild-type and CDK5 knockout samples were subjected to SDS-PAGE. ab40773 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/2000 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-CDK5 antibody [EP715Y] (ab40773) at 1/2000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

CDK5 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

SHSY5Y whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 33 kDa

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