Anti-CDKN2A/p16INK4a antibody [EPR20418]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CDKN2A/p16INK4a antibody [EPR20418] (AB211542)

Immunofluorescent analysis of 100% methanol-fixed MEF (mouse embryonic fibroblast cell line) and NIH/3T3 (mouse embyro fibroblast cell line) cells labeling CDKN2A/p16INK4a with ab211542 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1,000 dilution (green). Confocal image showing nuclear and cytoplasmic staining on MEF cell line.

Negative control : NIH/3T3 (PMID : 15210712).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1,000 dilution.

MEF cells were kindly provided by professor Pinlong Xu, Zhejiang University.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CDKN2A/p16INK4a antibody [EPR20418] (AB211542)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed MEF (mouse embryonic fibroblast cell line) cell line labeling CDKN2A/p16INK4awith ab211542 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

MEF cells were kindly provided by professor Pinlong Xu, Zhejiang University.

• IP

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Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR20418] (AB211542)

CDKN2A/p16INK4a was immunoprecipitated from fresh MEF lysates with ab211542 at 1/30 dilution (2ug in 0.35mg lysates). Lysates were made fresh and used immediately to minimize protein degradation. Western blot was performed on the immunoprecipitate using ab211542 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution. Lane 1 : MEF (mouse embryo fibroblast) whole cell lysate 10 μg (IP lysis buffer) Lane 2 : ab211542 in MEF (mouse embryo fibroblast) whole cell lysate (IP lysis buffer) Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab211542 in MEF whole cell lysate (IP lysis buffer) Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR20418] (ab211542)

All lanes:

MEF lysates

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 137s

• IP

Supplier Data

Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR20418] (AB211542)

CDKN2A/p16INK4a was immunoprecipitated from 0.35 mg of MEF (mouse embryonic fibroblast cell line) whole cell lysate with ab211542 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab211542 at 1/5000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1 : MEF whole cell lysate 10 μg (Input).

Lane 2 : ab211542 IP in MEF whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab211542 in MEF whole cell lysate.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 30 seconds.

MEF cells were kindly provided by professor Pinlong Xu, Zhejiang University.

All lanes:

Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR20418] (ab211542)

Predicted band size: 17 kDa

false

• WB

Supplier Data

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR20418] (AB211542)

Blocking/Dilution buffer : 5% NFDM/TBST.

Negative control : NIH/3T3 (PMID : 15210712).

Limited expression in mouse normal tissues has been documented in the literature (PMID : 9244355).

14 kDa band is a proteolytic p16INK4a lacking C-terminus (PMID : 18053084).

MEF cells are kindly provided by professor Pinlong Xu, Zhejiang University.

All lanes:

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR20418] (ab211542) at 1/2000 dilution

Lane 1:

MEF (mouse embryonic fibroblast cell line) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg

Lane 3:

Mouse lung lysate at 20 µg

Lane 4:

Mouse spleen lysate at 20 µg

Lane 5:

Mouse testis lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 17 kDa

Observed band size: 14 kDa,16 kDa

true

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR20418] (AB211542)

Blocking and dilution buffer : 5% NFDM/TBST.

ab181602 was used as loading control at 1/1000000 dilution.

All lanes:

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR20418] (ab211542) at 1/2000 dilution

Lane 1:

Mouse skin tissue lysate at 20 µg

Lane 2:

Mouse skin cancer tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 17 kDa

Observed band size: 14 kDa,16 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR20418] (AB211542)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR20418] (ab211542) at 1/1000 dilution

Lane 1:

His-tagged mouse CDKN2A/p16INK4a recombinant protein at 0.01 µg

Lane 2:

His-tagged mouse CDKN2B/p15INK4b recombinant protein at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 17 kDa

Observed band size: 18 kDa

true

Exposure time: 1s