Anti-PPP2R5D antibody [EPR15617-50] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PPP2R5D antibody [EPR15617-50] - BSA and Azide free (AB250953)

This data was developed using the same antibody clone in a different buffer formulation (ab188325) ab188325 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 ab188325 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PPP2R5D antibody [EPR15617-50] - BSA and Azide free (AB250953)

This data was developed using ab188325, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human brain tissue labeling PPP2R5D with ab188325 at 1/100 dilution, followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PPP2R5D antibody [EPR15617-50] - BSA and Azide free (AB250953)

This data was developed using ab188325, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human astrocytoma tissue labeling PPP2R5D with ab188325 at 1/100 dilution, followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

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

• WB

Lab

Western blot - Anti-PPP2R5D antibody [EPR15617-50] - BSA and Azide free (AB250953)

This data was developed using the same antibody clone in a different buffer formulation (ab188325)

ab188325 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 ab188325 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 [EPR15617-50] (<a href='/en-us/products/primary-antibodies/ppp2r5d-antibody-epr15617-50-ab188325'>ab188325</a>) 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

false

• IP

Supplier Data

Immunoprecipitation - Anti-PPP2R5D antibody [EPR15617-50] - BSA and Azide free (AB250953)

This data was developed using ab188325, the same antibody clone in a different buffer formulation.

Western blot analysis of PPP2R5D in 293T cell lysate immunoprecipitated with ab188325 at 1/50 dilution. A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1500 was used as secondary antibody.

All lanes:

Immunoprecipitation - Anti-PPP2R5D antibody [EPR15617-50] (<a href='/en-us/products/primary-antibodies/ppp2r5d-antibody-epr15617-50-ab188325'>ab188325</a>)

Predicted band size: 70 kDa

false

• WB

Supplier Data

Western blot - Anti-PPP2R5D antibody [EPR15617-50] - BSA and Azide free (AB250953)

This data was developed using ab188325, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-PPP2R5D antibody [EPR15617-50] (<a href='/en-us/products/primary-antibodies/ppp2r5d-antibody-epr15617-50-ab188325'>ab188325</a>) at 1/50000 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

false

• WB

Lab

Western blot - Anti-PPP2R5D antibody [EPR15617-50] - BSA and Azide free (AB250953)

This data was developed using the same antibody clone in a different buffer formulation (ab188325).

Lanes 1 - 4 : Merged signal (red and green). Green - ab188325 observed at 60-65 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab188325 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 A431 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab188325 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 [EPR15617-50] (<a href='/en-us/products/primary-antibodies/ppp2r5d-antibody-epr15617-50-ab188325'>ab188325</a>) 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

false