Anti-PPP2R5D 抗体 [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 1/200 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.

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.

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.

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

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.

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.

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).