HA721322

Cyclin D1 Recombinant Rabbit Monoclonal Antibody [PD01-64]

cat.: HA721322

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IHC-P, IF-Cell, FC
Clonality:	Monoclonal
Clone number:	PD01-64

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
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: 34 kDa
Isotype:	IgG
Immunogen:	Recombinant protein within Human Cyclin D1 aa 200-295 (C terminal).
Positive control:	MCF7 cell lysate, K-562 cell lysate, A431 cell lysate, Neuro-2a cell lysate, NIH/3T3 cell lysate, C6 cell lysate, SH-SY5Y cell lysate, Neuro-2a, MCF7, human colon carcinoma tissue, human testis tissue, NIH/3T3.
Subcellular location:	Cytoplasm, Nucleus, Membrane, Mitochondrion
Recommended Dilutions: WB IHC-P IF-Cell FC	1:5,000 1:200 1:2,000 1:5,000
Uniprot #:	SwissProt: P24385 Human \| P25322 Mouse \| P39948 Rat
Alternative names:	AI327039 B cell CLL/lymphoma 1 B cell leukemia 1 B cell lymphoma 1 protein B-cell lymphoma 1 protein BCL 1 BCL-1 BCL-1 oncogene BCL1 BCL1 oncogene ccnd1 cyclind1 CCND1/FSTL3 fusion gene CCND1/FSTL3 fusion gene, included CCND1/IGHG1 fusion gene CCND1/IGHG1 fusion gene, included CCND1/IGLC1 fusion gene CCND1/IGLC1 fusion gene, included CCND1/PTH fusion gene CCND1/PTH fusion gene, included CCND1_HUMAN cD1 Cyl 1 D11S287E G1/S specific cyclin D1 G1/S-specific cyclin-D1 Parathyroid adenomatosis 1 PRAD1 PRAD1 oncogene U21B31

Images

Fig1: Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody (HA721322) at 1/5,000 dilution and competitor's antibody at 1/5,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: K-562 cell lysate (negative)
Lane 3: A431 cell lysate
Lane 4: Neuro-2a cell lysate
Lane 5: NIH/3T3 cell lysate
Lane 6: C6 cell lysate
Lane 7: SH-SY5Y cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 34 kDa
Observed band size: 35 kDa

Exposure time: 20 seconds;

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 (HA721322) at 1/5,000 dilution and competitor's antibody at 1/5,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig2: Immunocytochemistry analysis of Neuro-2a cells labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody (HA721322) at 1/2,000 dilution and competitor's antibody at 1/1,600 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 Rabbit anti-Cyclin D1 antibody (HA721322) at 1/2,000 dilution and competitor's antibody at 1/1,600 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

	Fig3: Flow cytometric analysis of MCF7 cells labeling Cyclin D1. Cells were fixed and permeabilized. Then stained with the primary antibody (HA721322, red) at 1/5,000 dilution and competitor's antibody (red) at 1/2,000 dilution, compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
	Fig4: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-Cyclin D1 antibody (HA721322) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (HA721322) at 1/200 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.
	Fig5: Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-Cyclin D1 antibody (HA721322) at 1/50 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (HA721322) at 1/50 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.

Fig6: Immunocytochemistry analysis of NIH/3T3 cells labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody (HA721322) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-Cyclin D1 antibody (HA721322) at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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.

Fig7: Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody (HA721322) at 1/5,000 dilution.

Lane 1: MCF7-si NT cell lysate
Lane 2: MCF7-si Cyclin D1 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 34 kDa
Observed band size: 35 kDa

Exposure time: 17 seconds;

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 (HA721322) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

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