Anti mouse bcl 2 antibody

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The Anti-mouse Bcl-2 antibody is a laboratory reagent that binds specifically to the Bcl-2 protein in mouse cells. Bcl-2 is a key regulator of apoptosis, or programmed cell death, in mice. This antibody can be used to detect and quantify Bcl-2 expression in mouse tissues or cell lines.

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10 protocols using anti mouse bcl 2 antibody

Western Blot Analysis of Apoptosis Markers

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Following a previously described method [20 (link),21 (link)], Bax, Bcl-2, and cytochrome c expression was measured by western blot analysis. Using protein lysis buffer, hippocampal samples were lysed and a colorimetric protein assay kit was used to detect protein concentrations (Bio-Rad, Hercules, CA, USA). After 30 μg of protein was separated on sodium dodecyl sulfate-polyacrylamide gels, the separated protein was transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). Anti-mouse β-actin antibody (1:1,000; Santa Cruz Biotechnology), anti-rabbit Bax antibody (Cell signaling Technology; 1:1,000), anti-mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology), and anti-rabbit cytochrome c antibody (1:1,000; Cell signaling Technology) were used as the primary antibodies. The secondary antibodies were horseradish peroxidase–conjugated anti-mouse antibodies (1:3,000; Vector Laboratories) for β-actin and Bcl-2, and horseradish peroxidase–conjugated anti-rabbit antibodies (1:5,000; Vector Laboratories) for Bax and cytochrome c. An enhanced chemiluminescence detection system (Amersham Pharmacia Biotech GmbH, Freiburg, Germany) was used for band detection.

Kim M., Kim T.W., Kim C.J., Shin M.S., Hong M., Park H.S, & Park S.S. (2019). Berberine Ameliorates Brain Inflammation in Poloxamer 407-Induced Hyperlipidemic Rats. International Neurourology Journal, 23(Suppl 2), S102-110.

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Quantitative Western Blotting Assay

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Western blotting was performed, as the previously described method (Baek and Kim, 2016 (link); Kim et al., 2017 ). Tissue samples harvested from the hippocampus were lysed in the protein lysis buffer. Protein concentration was measured using a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein of 40 μg was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). Anti-mouse β-actin antibody (1:2,000; Santa Cruz Biotechnology), anti-mouse Bax antibody (1:1,000; Santa Cruz Biotechnology), anti-mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibody. Horseradish peroxidase-conjugated anti-mouse antibodies (1:2,000; Santa Cruz Biotechnology) for β-actin, Bax, and Bcl-2 were used as the secondary antibody. Band detection was performed using as enhanced chemiluminescence detection system (Amersham Pharmacia Biotech GmbH, Freiburg, Germany).

Lee J.M., Ji E.S., Kim T.W., Kim C.J., Shin M.S., Lim B.V., Chung Y.R, & Cho Y.S. (2018). Treadmill exercise improves memory function by inhibiting hippocampal apoptosis in pilocarpine-induced epileptic rats. Journal of Exercise Rehabilitation, 14(5), 713-723.

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Immunofluorescence Analysis of Myocardial Apoptosis

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The heart tissues of the mice were collected for immunofluorescence staining 14 days after DOX injection. Then, the heart tissues, specifically the myocardial regions, were sectioned at 5 μm thickness. All animal procedures were approved by the Research Ethics Committees of Ewha Womans University and conducted in accordance with approved guidelines. After fixation in 4% paraformaldehyde (Biosesang Inc., Seongnam, Korea), the tissues were blocked in 10% normal goat serum and incubated overnight at 4 °C with anti-mouse Bcl-2 antibody (1:100; Santa Cruz) and anti-rabbit survivin antibody (1:400; CST, Danvers, MA, USA). Anti-mouse AlexaFluor 647 goat (Life Technologies, Carlsbad, CA, USA) and anti-rabbit AlexaFluor 568 goat antibodies (Life Technologies, Carlsbad, CA, USA) were used as the secondary antibodies. After nuclear counter-staining with DAPI, the tissue slides were mounted and visualized with the LSM 800 confocal microscope.

Lee J.Y., Chung J., Byun Y., Kim K.H., An S.H, & Kwon K. (2021). Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Protect Cardiomyocytes from Doxorubicin-Induced Cardiomyopathy by Upregulating Survivin Expression via the miR-199a-3p-Akt-Sp1/p53 Signaling Pathway. International Journal of Molecular Sciences, 22(13), 7102.

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Gastrocnemius Muscle Protein Expression

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According to the previous study [30 (link)], we conducted western blotting. Tissue samples obtained by harvesting of gastrocnemius muscle were lysed using protein lysis buffer. Colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA) was used for the measuring of protein concentration. Protein (20 μg) was separated on sodium dodecyl sulfate-polyacrylamide gels, then it was transferred to a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). For the primary antibodies, we used anti-mouse β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1,000), anti-mouse Bax antibody (Santa Cruz Biotechnology; 1:1,000), anti-mouse Bcl-2 antibody (Santa Cruz Biotechnology; 1:1,000), and anti-rabbit caspase-3 antibody (Cell signaling Technology, Danvers, MA, USA; 1:1,000). For the secondary antibodies, we used horseradish peroxidase-conjugated anti-mouse antibodies (Santa Cruz Biotechnology; 1:3,000) for β-actin, Bax, Bcl-2, and anti-rabbit antibody (Santa Cruz Biotechnology; 1:5,000) for caspase-3. We calculate the detected bands using enhanced chemiluminescence detection system (Amersham Pharmacia Biotech GmbH, Freiburg, Germany).

Park S.S., Park H.S., Jeong H., Kwak H.B., No M.H., Heo J.W., Yoo S.Z, & Kim T.W. (2019). Treadmill Exercise Ameliorates Chemotherapy-Induced Muscle Weakness and Central Fatigue by Enhancing Mitochondrial Function and Inhibiting Apoptosis. International Neurourology Journal, 23(Suppl 1), S32-39.

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Immunofluorescence Analysis of Myocardial Bcl2 and Survivin

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The hearts of the mice were collected for immunofluorescence staining at 14 days after the DOX injection. Then the hearts, specifically the myocardial regions, were sectioned at 5 μm thickness. All animal procedures were approved by the Research Ethics Committees of Ewha Womans University and conducted in accordance with approved guidelines. After fixation in 4% paraformaldehyde (Biosesang Inc., Seongnam, Korea), the tissues were blocked in 10% normal goat serum and incubated with anti-mouse Bcl2 antibody (1:100; Santa Cruz Biotechnology) and anti-rabbit survivin antibody (1:400; Cell Signaling Technology) overnight at 4 °C. Anti-mouse AlexaFluor 647 goat (Life Technologies, Carlsbad, CA, USA) and anti-rabbit AlexaFluor 568 goat antibodies (Life Technologies) were used as the secondary antibodies. After nuclear counter-staining with DAPI, the tissue slides were mounted and visualized with the LSM 800.

Yoon Lee J., Chung J., Hwa Kim K., Hyun An S., Yi J.E., Ae Kwon K, & Kwon K. (2019). Extracorporeal shock waves protect cardiomyocytes from doxorubicin-induced cardiomyopathy by upregulating survivin via the integrin-ILK-Akt-Sp1/p53 axis. Scientific Reports, 9, 12149.

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Molecular Profiling of Hippocampal Signaling

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Western blot for the expressions of Bax, Bcl-2, BDNF, TrkB, CREB, and p-CREB were performed, according to the previously described method (Kim et al., 2014 (link); Ko et al., 2009 (link)). The hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH, 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 1.5 mM MgCl₂·6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Hercules, CA, USA). Protein samples (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. The membranes were incubated with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 and then incubated overnight at 4°C with the following primary antibodies: mouse anti-β-actin antibody, mouse anti-Bcl-2 antibody, mouse anti-Bax antibody, rabbit anti-BDNF antibody, rabbit anti-TrkB antibody, rabbit anti-CREB antibody, rabbit anti-p-CREB antibody (1:1,000; Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 hr with attempt secondary antibodies (1:2,000; Vector Laboratories), and ban detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).

Ko I.G., Kim S.E., Hwang L., Jin J.J., Kim C.J., Kim B.K, & Kim H. (2018). Late starting treadmill exercise improves spatial leaning ability through suppressing CREP/BDNF/TrkB signaling pathway following traumatic brain injury in rats. Journal of Exercise Rehabilitation, 14(3), 327-334.

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Immunohistochemical Analysis of Bax and Bcl-2

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Sections were dewaxed and rehydrated by conventional methods. After quenching endogenous peroxidase with 3% hydrogen peroxide for 10 min and blocking with normal goat serum for 10 min at 37°C, sections were incubated with rabbit anti-Bax antibody (1:200) and mouse anti-Bcl-2 antibody (1:200) (both from Santa Cruz Biotechnology, Inc.) at 4°C overnight. After washing in PBS, the sections were incubated with FITC-conjugated anti-rabbit IgG (1:500) and Cy3 conjugated anti-mouse IgG (1:200) (both from Beijing Cowin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature in the dark. Images were captured at a magnification of ×200 for analysis. The mean fluorescence intensity (MFI) was measured, and expression levels of Bax and Bcl-2 were calculated as change of the percentage in MFI compared to the vehicle control mice.

HOU X.Q., WU D.W., ZHANG C.X., YAN R., YANG C., RONG C.P., ZHANG L., CHANG X., SU R.Y., ZHANG S.J., HE W.Q., QU Z., LI S., SU Z.R., CHEN Y.B., WANG Q, & FANG S.H. (2014). Bushen-Yizhi formula ameliorates cognition deficits and attenuates oxidative stress-related neuronal apoptosis in scopolamine-induced senescence in mice. International Journal of Molecular Medicine, 34(2), 429-439.

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Protein Quantification and Western Blot Analysis

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The total protein extract for Bcl-2 and Bax quantification was prepared as indicated above and the equal volume of proteins (40 μg per lane) was resolved on 10% sodium dodecyl sulfate-polyacrylamide gel and then electrotransferred onto a polyvinylidene dfluoride (PVDF) membrane (Sigma; MO, USA). The membrane was blocked with TBS (Tris-buffered saline) which containing 0.3% Tween 20, 5% nonfat milk and 150 mM sodium chloride were incubated with primary antibody including mouse anti-Bcl-2 antibody, anti-Bax antibody (1:500; Santa Cruz Biotechnology, CA, USA) or rabbit monoclonal anti-rat β-actin (1:500; Zhongshan Biotechnology, Beijing) at 4°C for overnight and then washed with TBS to remove unbound antibodies. A secondary antibody was probed with horseradish peroxidase (HRP) linked anti-mouse antibody (1:1000; Santa Cruz Biotechnology, CA, USA) in TBS at room temperature for 1h and washed with TBS. The conjugated antibodies were detected by the enhanced chemiluminescence (Pierce; IL, USA) system, and the protein expression (band) are quantified using Bio-Rad image analyzer (GS-700 digital densitometer, GMI, Ramsey, MN 55303, USA). β-actin will serve as loading control.

Hou W.C., Miao X.H., Ma L.J., Bai X.X., Liu Q, & Song L. (2017). WITHAFERIN A INDUCES APOPTOSIS IN RAT C6 GLIOMA CELLS THROUGH REGULATING NF-KB NUCLEAR TRANSLOCATION AND ACTIVATION OF CASPASE CASCADE. African Journal of Traditional, Complementary, and Alternative Medicines, 14(2), 319-324.

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Monoclonal Antibody-Based HA Blocking Assay

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Monoclonal rat anti-CD44 antibody (Clone: 020; Isotype: IgG2b; obtained from CMB-TECH Inc., San Francisco, CA, USA) recognizes a determinant of the HA-binding region common to CD44 and its principal variant isoforms. This rat anti-CD44 was routinely used for HA-related blocking experiments. Immunoreagents such as rabbit anti-C-JUN antibody, mouse anti-Bcl-2 antibody and goat anti-actin antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-c-IAP-1 antibody, mouse anti-c-IAP-2 and mouse anti-XIAP antibody were from BD (Franklin Lakes, NJ, USA). Rabbit anti-phospho-c-Jun [pS63] antibody, rabbit anti-C-JUN antibody, rabbit anti-JNK[pS63] antibody and rabbit anti-JNK antibody were from Cell Signaling Technology (Beverly, MA, USA). JNK Inhibitor I, 420116 was purchased from EMD Millipore (Billerica, MA, USA). Doxorubicin hydrochloride was from Sigma Chemicals (St. Louis, MO). Healon HA polymers (~500,000-dalton polymers), purchased from Pharmacia & Upjohn Co. (Kalamazoo, MI), were prepared as described previously [29 (link),31 (link),38 (link)].

Chen L, & Bourguignon L.Y. (2014). Hyaluronan-CD44 interaction promotes c-Jun signaling and miRNA21 expression leading to Bcl-2 expression and chemoresistance in breast cancer cells. Molecular Cancer, 13, 52.

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Western Blot Analysis of Bax and Bcl-2

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For the detection of Bax and Bcl-2 expression, we used Western blotting, as the method described before [20 (link),21 (link)]. After separating 40-µg protein on sodium dodecyl sulfate-polyacrylamide gels, protein was transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). For the primary antibodies, we used mouse anti-actin antibody (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Bax antibody (1:1,000; Santa Cruz Biotechnology), and mouse antiBcl-2 antibody (1:1,000 Santa Cruz Biotechnology). As the secondary antibodies, we used mouse horseradish peroxidase-conjugated antibody (1:2,000; Santa Cruz Biotechnology) for actin, Bax, and Bcl-2. We used enhanced chemiluminescence detection system (Amersham Pharmacia Biotechnology GmbH, Freiburg, Germany) for the band detection. By Image-Pro Plus software (Media Cybernetics Inc.), the detected bands were densitometrically analyzed.

Lee J.M., Kim T.W., Park S.S., Han J.H., Shin M.S., Lim B.V., Kim S.H., Baek S.S., Cho Y.S, & Kim K.H. (2018). Treadmill Exercise Improves Motor Function by Suppressing Purkinje Cell Loss in Parkinson Disease Rats. International Neurourology Journal, 22(Suppl 3), S147-155.

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