HA721235

Bcl-2 Recombinant Rabbit Monoclonal Antibody [PD01-41]

cat.: HA721235

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human
Applications:	WB, IHC-P, IF-Cell, FC
Clonality:	Monoclonal
Clone number:	PD01-41

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: 26 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within Human Bcl2 alpha aa 50-150 (N terminal).
Positive control:	MCF7 cell lysate, Jurkat cell lysate, HL-60 cell lysate, THP-1 cell lysate, human B-cell lymphoma tissue, human tonsil tissue, human lymph nodes tissue, HeLa, THP-1.
Subcellular location:	Mitochondrion outer membrane, Nucleus membrane, Endoplasmic reticulum membrane, Cytoplasm.
Recommended Dilutions: WB IHC-P IF-Cell FC	1:2,000-1:5,000 1:1,000-1:5,000 1:100 1:1,000
Uniprot #:	SwissProt: P10415 Human
Alternative names:	Apoptosis regulator Bcl 2 Apoptosis regulator Bcl-2 Apoptosis regulator Bcl2 AW986256 B cell CLL/lymphoma 2 B cell leukemia/lymphoma 2 Bcl-2 Bcl2 BCL2_HUMAN C430015F12Rik D630044D05Rik D830018M01Rik Leukemia/lymphoma, B-cell, 2 Oncogene B-cell leukemia 2 PPP1R50 Protein phosphatase 1, regulatory subunit 50 Bcl 2

Images

Fig1: Western blot analysis of Bcl-2 on different lysates with Rabbit anti-Bcl-2 antibody (HA721235) at 1/2,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: MCF7 cell lysate (15 µg/Lane)
Lane 2: Jurkat cell lysate (15 µg/Lane)
Lane 3: HL-60 cell lysate (15 µg/Lane)
Lane 4: THP-1 cell lysate (15 µg/Lane)

Predicted band size: 26 kDa
Observed band size: 25 kDa

Exposure time: 1 minute 30 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 (HA721235) at 1/2,000 dilution and competitor's antibody at 1/2,000 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: Western blot analysis of Bcl-2 on different lysates with Rabbit anti-Bcl-2 antibody (HA721235) at 1/1,000 dilution.

Lane 1: Hela-si NT cell lysate (10 µg/Lane)
Lane 2: Hela-si Bcl-2 cell lysate (10 µg/Lane)

Predicted band size: 26 kDa
Observed band size: 26 kDa

Exposure time: 31 seconds;
ECL: merk

4-20% SDS-PAGE gel.

HA721235 was shown to specifically react with Bcl-2 in Hela-si NT cells. Weakened band was observed when Hela-si Bcl-2 sample was tested. Hela-si NT and Hela-si Bcl-2 samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (HA721235, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-rabbit IgG-HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

	Fig3: Immunohistochemical analysis of paraffin-embedded human B-cell lymphoma tissue with Rabbit anti-Bcl-2 antibody (HA721235) at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (HA721235) at 1/1,000 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.
	Fig4: Immunohistochemical analysis of paraffin-embedded human tonsil tissue with Rabbit anti-Bcl-2 antibody (HA721235) at 1/2,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (HA721235) at 1/2,000 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 lymph nodes tissue with Rabbit anti-Bcl-2 antibody (HA721235) at 1/5,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (HA721235) at 1/5,000 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 HeLa cells labeling Bcl-2 with Rabbit anti-Bcl-2 antibody (HA721235) 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 Rabbit anti-Bcl-2 antibody (HA721235) at 1/100 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.
	Fig7: Immunocytochemistry analysis of THP-1 cells labeling Bcl-2 with Rabbit anti-Bcl-2 antibody (HA721235) 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 Rabbit anti-Bcl-2 antibody (HA721235) at 1/100 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.
	Fig8: Flow cytometric analysis of HeLa cells labeling Bcl-2. Cells were fixed and permeabilized. Then stained with the primary antibody (HA721235, 1μg/mL) (red) 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).

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