Anti-PPP2R5D antibody [EPR15617]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PPP2R5D antibody [EPR15617] (AB188323)

ab188323 was shown to react with PPP2R5D in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a PPP2R5D knockout cell line. 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 ab188323 at 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 ?g/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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PPP2R5D antibody [EPR15617] (AB188323)

Intracellular Flow Cytometry analysis of HeLa cells labeling PPP2R5D using ab188323 at 1/40 dilution (pink). A Goat anti rabbit IgG (FITC) at 1/150 dilution was used as secondary antibody. Cells were fixed with 2% paraformaldehyde. Isotype control : Rabbit monoclonal IgG (green).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PPP2R5D antibody [EPR15617] (AB188323)

Immunohistochemical analysis of paraffin-embedded Human brain tissue labeling PPP2R5D using ab188323 at 1/100 dilution. A Ready to use HRP Polymer for Rabbit IgG (prediluted) was used as secondary. Counterstain : Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PPP2R5D antibody [EPR15617] (AB188323)

Immunofluorescence analysis of HepG2 cells labeling PPP2R5D using ab188323 at 1/250 dilution. A Goat anti rabbit IgG (Alexa Fluor488) at 1/200 dilution was used as secondary antibody. Cells were fixed with 4% Paraformaldehyde. Counterstain : DAPI.

• WB

Lab

Western blot - Anti-PPP2R5D antibody [EPR15617] (AB188323)

ab188323 was shown to react with PPP2R5D in wild-type HAP1 cells in Western blot with loss of signal observed in a PPP2R5D knockout cell line. Wild-type HAP1 and PPP2R5D knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab188323 overnight at 4 °C at a 1/10000 dilution. Blots were incubated with secondary antibodies at 0.2ug/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-PPP2R5D antibody [EPR15617] (ab188323) at 1/10000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

PPP2R5D knock-out HAP1 lysate at 20 µg

Observed band size: 70 kDa

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• IP

Supplier Data

Immunoprecipitation - Anti-PPP2R5D antibody [EPR15617] (AB188323)

Immunoprecipitation analysis of 293T cell lysate labeling PPP2R5D using ab188323 at 1/50 dilution. A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1500 was used as secondary antibody.

All lanes:

Immunoprecipitation - Anti-PPP2R5D antibody [EPR15617] (ab188323)

Predicted band size: 70 kDa

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• WB

Supplier Data

Western blot - Anti-PPP2R5D antibody [EPR15617] (AB188323)

Based on the sequence analysis, ab188323 recognizes 3 isoforms with the predicted MWs of 58KDa, 66KDa and 70KDa, respectively.

All lanes:

Western blot - Anti-PPP2R5D antibody [EPR15617] (ab188323) at 1/100000 dilution

Lane 1:

C6 cell lysate at 10 µg

Lane 2:

HepG2 cell lysate at 10 µg

Lane 3:

293T cell lysate at 10 µg

Lane 4:

HeLa cell lysate at 10 µg

Lane 5:

Jurkat cell 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 - Anti-PPP2R5D antibody [EPR15617] (AB188323)

Lanes 1 - 4 : Merged signal (red and green). Green - ab188323 observed at 60-65 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab188323 was shown to react with PPP2R5D in wild-type A431 cells in western blot with loss of signal observed in PPP2R5D knockout sample. Wild-type and PPP2R5D knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab188323 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 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 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PPP2R5D antibody [EPR15617] (ab188323) at 1/10000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

PPP2R5D knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human PPP2R5D knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-ppp2r5d-knockout-a-431-cell-line-ab270476'>ab270476</a>)

Lane 3:

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

Lane 4:

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

Predicted band size: 70 kDa

Observed band size: 60-65 kDa

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