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 cytometric analysis of 4% paraformaldehyde-fixed MEF (mouse embryonic fibroblast cell line) cell line labeling CDKN2A/p16INK4a with 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.

All lanes : Anti-CDKN2A/p16INK4a antibody [EPR20418] (ab211542) at 1/1000 dilution

Lane 1 : His-tagged mouse CDKN2A/p16INK4a recombinant protein
Lane 2 : His-tagged mouse CDKN2B/p15INK4b recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 18 kDa
Observed band size: 18 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-CDKN2A/p16INK4a antibody [EPR20418] (ab211542) at 1/2000 dilution

Lane 1 : MEF (mouse embryonic fibroblast cell line) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
Lane 3 : Mouse lung lysate
Lane 4 : Mouse spleen lysate
Lane 5 : Mouse testis lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 18 kDa
Observed band size: 14,16 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

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.

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.