Western blot - Anti-p21 antibody [EPR18021] (ab188224)

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression level of p21 protein can be induced using staurosporine (protein kinase C inhibitor) (PMID:7677742).

All lanes:

Western blot - Anti-p21 antibody [EPR18021] (ab188224) at 1/1000 dilution

Lane 1:

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

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast cell line) treated with 1μM staurosporine for 2hrs, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 18 kDa

Observed band size: 18 kDa

Exposure time: 3min

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR18021] (ab188224)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling p21 with ab188224 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on mouse testis is observed (PMID: 9170103).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunocytochemistry/ Immunofluorescence - Anti-p21 antibody [EPR18021] (ab188224)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling p21 with ab188224 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on RAW264.7 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

Flow Cytometry (Intracellular) - Anti-p21 antibody [EPR18021] (ab188224)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling p21with ab188224 at 1/50 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Western blot - Anti-p21 antibody [EPR18021] (ab188224)

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes:

Western blot - Anti-p21 antibody [EPR18021] (ab188224) at 1/1000 dilution

All lanes:

RAW264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 18 kDa

Observed band size: 18 kDa

Exposure time: 8s

Immunoprecipitation - Anti-p21 antibody [EPR18021] (ab188224)

p21 was immunoprecipitated from 0.35 mg of NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate with ab188224 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab188224 at 1/500 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10 μg (Input).

Lane 2: ab188224 IP in NIH/3T3 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab188224 in NIH/3T3 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds.

All lanes:

Immunoprecipitation - Anti-p21 antibody [EPR18021] (ab188224)

Predicted band size: 18 kDa