HA601131

p16INK4a Recombinant Mouse Monoclonal Antibody [PD01-16]

cat.: HA601131

Product Type:	Recombinant Mouse monoclonal IgG1, primary antibodies
Species reactivity:	Human
Applications:	IHC-P, WB, IF-Cell, IF-Tissue
Clonality:	Monoclonal
Clone number:	PD01-16

Form:	Liquid
Storage condition:	Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
Storage buffer:	PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 16 kDa
Isotype:	IgG1
Immunogen:	Synthetic peptide within N terminal Human p16INK4a.
Positive control:	Human tonsil tissue, human cervix poorly differentiated adenocarcinoma tissue, human cervix highly differentiated squamous cell carcinoma tissue, human ovary Advanced serous carcinoma tissue, HeLa cell lysate, HepG2 cell lysate, HEK-293 cell lysate, NIH:OVCAR-3 cell lysate, HeLa.
Subcellular location:	Cytoplasm, Nucleus.
Recommended Dilutions: IHC-P WB IF-Cell IF-Tissue	1:200-1:500 1:1,000 1:100 1:500
Uniprot #:	SwissProt: P42771 Human
Alternative names:	ARF CDK4 inhibitor p16 INK4 CDK4I CDKN2 CDKN2A Cell cycle negative regulator beta CMM2 Cyclin dependent kinase 4 inhibitor A Cyclin dependent kinase inhibitor 2A INK4 INK4a Melanoma p16 inhibits CDK4 MLM MTS 1 MTS1 Multiple tumor suppressor 1 p16 p16 INK4a p16INK4a p19 P19ARF TP16

Images

	Fig1: Immunohistochemical analysis of paraffin-embedded human tonsil tissue with Mouse anti-p16INK4a antibody (HA601131) at 1/500 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA601131) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig2: Application: Immunohistochemistry (IHC-P) Species: Human Tissue: Cervix poorly differentiated adenocarcinoma Sample: Paraffin-embedded section Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃. Wash buffer: 1× TBST Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature. Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature. Primary antibody: HA601131, 1/500, 1 hour at room temperature. Secondary antibody: HA1120, 20 minutes at room temperature.
	Fig3: Application: Immunohistochemistry (IHC-P) Species: Human Tissue: Cervix well-differentiated squamous cell carcinoma Sample: Paraffin-embedded section Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃. Wash buffer: 1× TBST Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature. Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature. Primary antibody: HA601131, 1/200, 1 hour at room temperature. Secondary antibody: HA1120, 20 minutes at room temperature.

Fig4: Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: High-grade serous ovarian carcinoma
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA601131, 1/500, 1 hour at room temperature.
Secondary antibody: HA1120, 20 minutes at room temperature.

Fig5: Western blot analysis of p16INK4a on different lysates with Mouse anti-p16INK4a antibody (HA601131) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HepG2 cell lysate
Lane 3: HEK-293 cell lysate
Lane 4: NIH:OVCAR-3 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 16 kDa
Observed band size: 16 kDa

Exposure time: 2 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601131) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:100,000 dilution was used for 1 hour at room temperature.

	Fig6: Immunocytochemistry analysis of HeLa cells labeling p16INK4a with Mouse anti-p16INK4a antibody (HA601131) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-p16INK4a antibody (HA601131) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.
	Fig7: Immunocytochemistry analysis of HeLa cells labeling p16INK4a with Mouse anti-p16INK4a antibody (HA601131) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-p16INK4a antibody (HA601131) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Ki67 (HA721115, green) was stained at 1/250 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution.
	Fig8: Immunohistochemical analysis of paraffin-embedded human High-grade serous carcinoma tissue with Mouse anti-p16INK4a antibody (HA601131) at 1/200 dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. The section was incubated with HA601131 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

	Fig9: Application: Immunohistochemistry (IHC-P) Species: Human Tissue: High-grade serous ovarian carcinoma Sample: Paraffin-embedded section Primary antibody dilution: 1/200 Antigen retrieval: ER2 Platform: Leica Biosystems BOND® RX
	Fig10: Application: Immunohistochemistry (IHC-P) Species: Human Tissue: Cervix well-differentiated squamous cell carcinoma Sample: Paraffin-embedded section Primary antibody dilution: 1/200 Antigen retrieval: ER2 Platform: Leica Biosystems BOND® RX
	Fig11: Application: Immunohistochemistry (IHC-P) Species: Human Tissue: Ovary Advanced serous carcinoma Sample: Paraffin-embedded section Primary antibody dilution: 1/200 Antigen retrieval: HIER (CC1) Platform: Ventana® BenchMark ULTRA

Fig12: Application: Immunofluorescence (IF-tissue)

Species: Human
Tissue: Tonsil
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

Wash buffer: 1× PBS
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA601131, 1/500, overnight at 4℃.
Secondary antibody: Goat Anti-Mouse IgG (iFluor™ 488, HA1125), 1.5 hours at room temperature.

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.