Figure 1: CDKN2A/p16INK4a target protein structure.

CDKN2A/p16INK4a target introduction

Protein function

• P16INK4a (p16) is a cyclin-dependent kinase inhibitor that blocks the activation of the E2F transcription factor by inhibiting CDK4/6, negatively regulating the proliferation of normal cells.
• Members of the INK4 cyclin-dependent kinase inhibitor family include p16 INK4A, p15 INK4B, p18 INK4C, and p19 INK4D.
• CDKN2A/p16INK4a is located on chromosome 9p21.3 and is adjacent to the tumor suppressor gene cluster that is lost.
• CDKN2A/p16INK4a affects tissue homeostasis and maintains a coordinated balance between tumor suppression and aging.
• Although ARF and p16INK4A (Q8N726) are encoded by the same gene CDKN2A, they are completely different proteins.

Protein expression

• It is hardly expressed in normal tissues. It is only expressed in some tumor tissues, such as cervical cancer and breast cancer. In addition, it is only expressed in some cell lines.

Protein localization

• CDKN2A/p16INK4a is distributed in the cytoplasm and nucleus of cells.

Figure 2: ICC experimental results of the CDKN2A/p16INK4a target. Anti-CDKN2A/p16INK4a [EPR20418] antibody product (ab211542). Green: CDKN2A/p16INK4a, Red: Tubulin, Blue: DAPI.

Isoforms & Post-translation modifications

• Human (P42771): Isoforms 1-3, 5: 11-18 kDa (predicted)
• Mouse (P51480): Isoforms 1-2: 13-18 kDa (predicted)
• Rat (Q9R0Z3): 17.4 kDa (predicted)
• Phosphorylation enhances its interaction with CDK4.
• Acetylation.

WB experiment tips

Precautions

• The expression of CDKN2A/p16INK4a varies in different tissue cells. It is hardly expressed in normal tissues (such as brain tissue, skeletal muscle tissue, etc.) but mainly expressed in some tumor tissues, such as cervical cancer, breast cancer, etc.
• It is only expressed in some cell lines. We strongly recommend adding positive controls (HeLa, HEK-293T, etc.) to confirm that the experimental system works appropriately.
• CDKN2A/p16INK4a is a small protein with a molecular weight of 17 kDa. We recommend using a 15% SDS-PAGE gel or a concentration gradient gel for electrophoresis in WB experiments to obtain ideal experimental results. Improper selection of PVDF membrane pore size and excessive transfer time may result in no signal or weak signal.
• CDKN2A/p16INK4a has multiple isoforms with molecular weights ranging from 11 to 18 kDa, and multiple band phenomena may be detected in WB experiments (as shown in Figure 4).
• Do not cut the membrane to prevent loss of the target protein band.

Positive controls

• HeLa, HEK-293T whole cell lysate
• Recombinant human CDKN2A/p16INK4a protein (Tagged) (ab268831)

Negative controls (no or weak expression)

• MCF7 whole cell lysate
• MDA-MB-231 whole cell lysate
• NIH/3T3 whole cell lysate
• Mouse lung tissue
• Brain tissue

Example of results

Figure 3: Western blot - Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal (ab108349).

Primary antibody: Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal (ab108349) at a dilution of 1/2000.
Blocking: 5% NFDM/TBST blocking solution.

Lane 1: HEK-293 whole cell lysate; Lane 2: Saos-2 whole cell lysate.

Predicted band size: 17 kDa
Detected band size: 17 kDa

Figure 4: WB-Anti-CDKN2A antibody [EPR24167-43] antibody product (ab270058).

Primary antibody: Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (ab270058) was used at a dilution of 1/1000.
Blocking: 5% NFDM/TBST blocking solution was used.

Lane 1: HeLa whole cell lysate;
Lane 2: HEK-293 whole cell lysate;
Lane 3: Saos-2 whole cell lysate;
Lane 4: MCF7 whole cell lysate;
Lane 5: MDA-MB-231 whole cell lysate.

Predicted band size: 17 kDa
Detected band size: 16 kDa

Figure 5: CDKN2A/p16INK4a is expressed differently in various tissues of mice and almost not expressed in normal tissues. (PMID: 9244355)

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Use a complex proteinase inhibitor to prevent degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.
4. Set positive controls (such as HeLa, HEK-293T) and negative controls (MCF7 whole cell lysate).

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.
2. For target proteins with smaller molecular weights, it is recommended to use a 15% concentration separation gel for electrophoresis.

Transfer:

1. We recommend using a 0.22 μm PVDF membrane.
2. We strongly recommend using Ponceau S staining after transfer to confirm the success of the transfer.

References

1. Sherr, C., Beach, D. and Shapiro, G., 2015. Targeting CDK4 and CDK6: From Discovery to Therapy. Cancer Discovery, 6(4), pp.353-367.
2. LaPak, K. and Burd, C., 2013. The Molecular Balancing Act of p16INK4a in Cancer and Aging. Molecular Cancer Research, 12(2), pp.167-183.
3. Yang, D., Liu, L. and Zheng, X., 2008. Cyclin-dependent kinase inhibitor p16INK4a and telomerase may co-modulate endothelial progenitor cells senescence. Ageing Research Reviews, 7(2), pp.137-146.
4. C Romagosa, S Simonetti, L López-Vicente, A Mazo, M E Lleonart, J Castellvi, S Ramon y Cajal. p16(Ink4a) overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors. Oncogene. 2011 May 5;30(18):2087-97. doi: 10.1038/onc.2010.614. Epub 2011 Feb 7.