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Anti cdkn2a p16ink4a antibody

Manufactured by Abcam
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The Anti-CDKN2A/p16INK4a antibody is a laboratory reagent used for the detection and study of the CDKN2A/p16INK4a protein, which is a key regulator of the cell cycle. This antibody can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to identify and quantify the expression of the CDKN2A/p16INK4a protein in biological samples.

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Lab products found in correlation

Cyclin a, by Santa Cruz Biotechnology (2 mentions) Enhanced chemiluminescence kit, by Thermo Fisher Scientific (2 mentions) Pcdk2 t160, by Cell Signaling Technology (2 mentions) Perk y204 sc 7383, by Santa Cruz Biotechnology (2 mentions) Phospho cdk4 pa5 664482 antibody, by Thermo Fisher Scientific (2 mentions) Cyclin e1, by Cell Signaling Technology (2 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions) Halt protease inhibitor, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Santa Cruz Biotechnology (2 mentions) Normal horse serum, by Thermo Fisher Scientific (1 mentions) Trueseq stranded total rna sample prep kit, by Illumina (1 mentions) Casava 1, by Illumina (1 mentions) Rneasy plus kit, by Qiagen (1 mentions) Hiseq 3000, by Illumina (1 mentions) Model eclipse ni u, by Nikon (1 mentions) Vectashield plus antifade mounting medium, by Vector Laboratories (1 mentions) Goat anti mouse igg, by Abcam (1 mentions)

5 protocols using anti cdkn2a p16ink4a antibody

1

Immunoblot Analysis of Cell Signaling

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Primary antibodies for immunoblot analysis that were purchased from Cell Signaling Technology include: p-RB (S807/S811) (8516S), RB (9313S) (p-Akt (S473) (4070S), Akt (4691), pS6 (S235/236) (2211), S6 (2217), cyclin E1 (4129S), p21 (2947S), p27 (2552S), CDK4 (12790), Raptor (2280S), Rictor (2140S), pCDK2 (T160) (2561S) and CDK2 (2546S). Antibodies from Santa Cruz Biotechnology include: actin (SC-47778), pERK (Y204) (SC-7383), ERK (SC-514302), cyclin D1 (SC20044), CDK2 (SC-6248) and cyclin A (SC-271682). Phospho-CDK4 (PA5-664482) antibody was purchased from Thermo Fisher. Anti-CDKN2A/p16INK4a antibody was purchased from Abcam. The whole-cell extracts were prepared by lysing the cells with RIPA lysis buffer (Santa Cruz Biotechnology, SC-24948A) in the presence of 1X Halt protease inhibitor (Thermo Fisher) and 1 mM PMSF (Sigma). The extracted proteins (20 μg) were resolved by SDS-PAGE and transferred to PVDF membranes, which were then incubated with primary antibodies at 4°C overnight, followed by incubation with HRP tagged anti-mouse or anti-rabbit secondary antibodies at room temperature up to 1 hour. An enhanced chemiluminescence kit (Thermo Fisher, 34076) was used to detect the immuno-reactive bands.
Knudsen E.S., Kumarasamy V., Ruiz A., Sivinski J., Chung S., Grant A., Vail P., Chauhan S.S., Jie T., Riall T.S, & Witkiewicz A.K. (2019). Cell cycle plasticity driven by MTOR signaling: Integral resistance to CDK4/6 inhibition in patient-derived models of pancreatic cancer. Oncogene, 38(18), 3355-3370.
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2

Senescence-Associated Beta-Galactosidase and p16 Staining

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Cells were grown on coverslips and stained for SA-βgal as described above. Immunocytofluorescence staining for p16 was performed subsequently. Cells were incubated with 10% Triton X-100 (Amreco) for 3 min and washed with PBS. Nonspecific binding was blocked by incubating with 1% normal horse serum (Gibco) at 37°C for 1 h. Cells were then incubated with anti-CDKN2A/p16INK4a antibody (Abcam, ab108349) (1:10,000) at 4°C overnight, followed by incubation with secondary antibody Alexa Fluor 488 conjugate goat anti-rabbit IgG (1:10,000) (Cell Signaling Technology) in the dark at 37°C for 30 min. The coverslip with stained cells was washed and mounted using Fluoroshield mounting medium and DAPI (Abcam, ab104139). SA-βgal and p16 positive cells were visualized and imaged using an EVOS FL Auto 2 Cell Imaging System.
Chuenwisad K., More-krong P., Tubsaeng P., Chotechuang N., Srisa-Art M., Storer R.J, & Boonla C. (2021). Premature Senescence and Telomere Shortening Induced by Oxidative Stress From Oxalate, Calcium Oxalate Monohydrate, and Urine From Patients With Calcium Oxalate Nephrolithiasis. Frontiers in Immunology, 12, 696486.
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3

Immunoblot Analysis of Cell Signaling

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Primary antibodies for immunoblot analysis that were purchased from Cell Signaling Technology include: p-RB (S807/S811) (8516S), RB (9313S) (p-Akt (S473) (4070S), Akt (4691), pS6 (S235/236) (2211), S6 (2217), cyclin E1 (4129S), p21 (2947S), p27 (2552S), CDK4 (12790), Raptor (2280S), Rictor (2140S), pCDK2 (T160) (2561S) and CDK2 (2546S). Antibodies from Santa Cruz Biotechnology include: actin (SC-47778), pERK (Y204) (SC-7383), ERK (SC-514302), cyclin D1 (SC20044), CDK2 (SC-6248) and cyclin A (SC-271682). Phospho-CDK4 (PA5-664482) antibody was purchased from Thermo Fisher. Anti-CDKN2A/p16INK4a antibody was purchased from Abcam. The whole-cell extracts were prepared by lysing the cells with RIPA lysis buffer (Santa Cruz Biotechnology, SC-24948A) in the presence of 1X Halt protease inhibitor (Thermo Fisher) and 1 mM PMSF (Sigma). The extracted proteins (20 μg) were resolved by SDS-PAGE and transferred to PVDF membranes, which were then incubated with primary antibodies at 4°C overnight, followed by incubation with HRP tagged anti-mouse or anti-rabbit secondary antibodies at room temperature up to 1 hour. An enhanced chemiluminescence kit (Thermo Fisher, 34076) was used to detect the immuno-reactive bands.
Knudsen E.S., Kumarasamy V., Ruiz A., Sivinski J., Chung S., Grant A., Vail P., Chauhan S.S., Jie T., Riall T.S, & Witkiewicz A.K. (2019). Cell cycle plasticity driven by MTOR signaling: Integral resistance to CDK4/6 inhibition in patient-derived models of pancreatic cancer. Oncogene, 38(18), 3355-3370.
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4

Comprehensive RNA and Protein Expression Profiling

Cited 4 times
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Whole transcriptome RNA expression was determined by RNASeq. Briefly, cell line RNA was isolated with an RNeasy Plus kit (Qiagen) and Illumina compatible libraries made with a TrueSeq Stranded Total RNA Sample Prep Kit (Illumina, Inc.). Libraries were sequenced with an Illumina HiSeq3000 instrument using the 75-bp paired-end format. Post sequencing, BCL files were converted to “FastQ.gz” format and individual sample libraries de-multiplexed using CASAVA 1.8.2 software (Illumina) with no mismatches. FASTQ files were checked for read quality using FastQC, aligned to hg19 using TopHat [16 (link)], and alignment quality was checked with RSeQC [17 (link)]. BAM files with mapped reads were sorted and outputted with SAMtools [18 (link)], and reads mapping to genes were counted with HTSeq-count [19 (link)]. Reads were then normalized using the trimmed mean of M (TMM) method implemented in the R Bioconductor package edgeR to generate expression values for each gene [20 (link)]. Protein expression for p16 was determined following Reverse Phase Protein Array analysis of cell lysates as previously described [21 (link)], using anti-CDKN2A/p16INK4a antibody (catalogue # ab81278) from Abcam.
Gadhikar M.A., Zhang J., Shen L., Rao X., Wang J., Zhao M., Kalu N.N., Johnson F.M., Byers L.A., Heymach J., Hittelman W.N., Udayakumar D., Pandita R.K., Pandita T.K., Pickering C.R., Redwood A.B., Piwnica-Worms H., Schlacher K., Frederick M.J, & Myers J.N. (2017). CDKN2A/p16 deletion in head and neck cancer cells is associated with Cdk2 activation, replication stress, and vulnerability to Chk1 inhibition. Cancer research, 78(3), 781-797.
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5

Immunohistochemical Analysis of p16Ink4a Expression

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Liver was fixed overnight in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). After being embedded in paraffin, the specimens were cut cross-sectionally into 4-µm sections. All sections were incubated in sodium citrate solution (pH 6.0) at 95 °C for 40 min and then blocked with 5% fetal bovine serum in PBS containing 0.1% Tween-20 at room temperature for 2 h. Sections were then incubated with anti-CDKN2A/p16Ink4a antibody (catalog no. 54210; Abcam, Waltham, MA, USA) with 1:250 dilution at 4 °C for overnight in moist chamber. They were then washed and incubated with goat anti-mouse IgG (catalog no. ab150116; Abcam, Waltham, MA, USA) with 1:500 dilution at room temperature for 1 h. Vectashield Plus antifade mounting medium (catalog no. H-1900; Vector Laboratories, Newark, CA, USA) containing DAPI at 1:5,000 dilution was applied, and fluorescent signals were visualized under a fluorescent microscope (Ni-U model eclipse, Nikon, Japan).
Somsura R., Kamkajon K., Chaimongkolnukul K., Chantip S., Teerapornpuntakit J., Wongdee K., Kamonsutthipaijit N., Tangtrongsup S., Panupinthu N., Tiyasatkulkovit W, & Charoenphandhu N. (2023). Tissue-specific expression of senescence biomarkers in spontaneously hypertensive rats: evidence of premature aging in hypertension. PeerJ, 11, e16300.
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