Anti-microtubule associated protein 1 light chain 3 (LC3) (NB100-2220) and anti-Beclin 1 (ab55878) antibodies were purchased from Novus Biological (LLC, USA). Anti-Bcl-2-associated X protein (Bax) and anti-Bcl-2 antibodies were purchased from Abcam (Cambridge, MA, USA). Antibodies recognizing phospho-mTOR (Ser2448), mTOR, phospho-4E-BP1 (Thr37/46), 4E-BP1, phospho-p85S6K (Thr412), p85S6K, phospho-p70S6K (Thr389), and p70S6K were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipofectamine 2000 (11668-019), LysoTracker Red (L7528) and MitoTracker (M7512) were purchased from Invitrogen. The β-actin (KC-5A08) and GAPDH (KC-5G5) antibodies were from Kangcheng (Shanghai, China). Melatonin and all other reagents were purchased from Sigma Chemical Co. (St. Louis, Missouri) unless otherwise indicated.
Anti bcl 2 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-Bcl-2 antibody is a laboratory research tool used for the detection of the Bcl-2 protein in various biological samples. Bcl-2 is a key regulator of apoptosis, and the antibody can be used to study its expression and localization.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax antibody, by Abcam (15 mentions)
Anti gapdh antibody, by Abcam (7 mentions)
Anti caspase 3 antibody, by Abcam (6 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti β actin antibody, by Abcam (4 mentions)
Anti cleaved caspase 3 antibody, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti cyclin b1, by Abcam (2 mentions)
Anti cyclin a2, by Abcam (2 mentions)
Anti bax antibody, by Proteintech (2 mentions)
Hrp goat anti mouse anti rabbit igg, by Proteintech (2 mentions)
Anti ndrg2, by Proteintech (2 mentions)
Anti β actin 60008 1 ig, by Proteintech (2 mentions)
Ripa lysis buffer, by Applygen (2 mentions)
Anti p erk5 antibody, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Lc3 nb100 2220, by Novus Biologicals (1 mentions)
40 protocols using anti bcl 2 antibody
1
Autophagy markers and signaling proteins
2
Immunoblot Analysis of Apoptosis Regulators
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HeLa and SiHa cells, transfected with shTCONS_00026907 (shRNA and NC), and the treated BALB/c athymic nude mice tissue samples were lysed using a lysis buffer, including a protease inhibitor cocktail (Sigma‐Aldrich, USA). The proteins in equal concentrations were separated by 8% SDS/PAGE gels and transferred to PVDF membranes (Millipore, USA). The primary antibodies were used to incubate the PVDF membranes with the target proteins at 4°C overnight. The HRP‐conjugated secondary antibodies were used to incubate the treated PVDF membranes for 1 h at room temperature. The primary antibodies used in our study were the anti‐ELK‐1 (1:1000, Santa Cruz Biotechnology, USA)), the anti‐p‐ELK1 (1:1000, Santa Cruz Biotechnology, USA), the anti‐C‐Fos (1:1000, Santa Cruz Biotechnology, USA), the anti‐Cyclin D1 (1:1000, Beijing Genomics Institute, China), and the anti‐Bcl‐2 antibodies (1:200; Abcam, USA). The anti‐GAPDH antibody (1:4000, Beverly, USA) was used as an internal control.
3
Western Blot Analysis of IGFBP5, ERK, and Apoptosis Markers
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According to standard methods, Western blot analysis was performed. Briefly, proteins were separated on 10% SDS‐PAGE gels and then transferred to PVDF membranes (Amersham). The membranes were blocked using 5% nonfat dried milk for 2 hours and then incubated for 12 hours with an anti‐IGFBP5 antibody (1:1000; Abcam), anti‐ERK antibody (1:1000; Abcam), anti‐pERK (phosphorylated ERK) antibody (1:500; Abcam), anti‐Bax antibody (1:1000; Abcam), anti‐caspase‐3 antibody (1:1000; Abcam), anti‐Bcl2 antibody (1:1000; Abcam) or anti‐GAPDH antibody (1:10 000; Abcam). After washing in TBST (10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl and 0.1% Tween 20), the membranes were incubated for 2 hours with a goat anti‐rabbit antibody (1:5000; Abcam). Normalization was performed by blotting the same membranes with an antibody against GAPDH.
4
Protein Extraction and Western Blotting
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The proteins from cells were extracted using RIPA Lysis Buffer (10 μl PMSF [100 mM] was added to 1ml RIPA Lysis Buffer). Two hundred microliters of RIPA Lysis Buffer was added to each culture dish and lysed on ice for 30 min. Then, the sample was centrifuged at 4°C for 10 min (16,000 g ). The supernatant was stored at −20°C. The protein concentration of each sample was measured by BCA Protein Quantitative Kit (Invitrogen, U.S.A.) following to the manufacturer’s recommendations. The proteins were separated by SDS/PAGE and transferred to nitrocellulose membrane (Thermo Fisher Scientific). The membranes were blocked in 5% skim milk for 1–2 h, then incubated with anti-RAC3 antibody, anti-BCL-2 antibody, anti-PML antibody, and anti-β-actin antibody (all at 1:1000 dilutions; Abcam, U.K.) overnight at 4°C. Finally, the membranes were incubated with secondary antibodies at room temperature for 1–2 h, and developed by ECI (Thermo Fisher Scientific).
5
Western Blot Analysis of Protein Expression
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RIPA lysis buffer (Thermo Fisher Scientific, Inc.) was used to extract total protein from the cultured cells. Subsequently, protein concentration was determined using an Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). The proteins (20 µg/lane) were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore). Then 5% milk PBS with 0.1% Triton X-100 was applied to block the membranes at room temperature for 2 h, which were then incubated with the following: Anti-HMGA2 antibody (cat. no. ab97276; 1:2,000; Abcam), anti-XIAP antibody (cat. no. ab21278; 1:1,000; Abcam), anti-Bcl-2 antibody (cat. no. ab32124; 1:1,000; Abcam), anti-cleaved caspase-3 antibody (cat. no. ab2302; 1:1,000; Abcam), anti-Wnt antibody (cat. no. ab28472; 1:1,000; Abcam), anti-non-phospho (Np)-β-catenin antibody (cat. no. 8814; 1:1,000; Cell Signaling Technology, Inc.) and anti-GAPDH antibody (cat. no. ab9485; 1:2,500; Abcam) overnight at 4°C. The membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. SA00001-2; ProteinTech Group, Inc.) at 4°C for 1 h after washing with PBST (containing 0.05% Tween-20) three times. Protein bands were detected with ECL (Thermo Fisher Scientific, Inc.) and visualized using Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc.).
6
Investigating Bcl-2 Interaction with Beclin1
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The chondrocytes were lysed with the cell lysis buffer containing protease inhibitor on ice for 30 minutes. The lysate was collected by centrifugation at 4°C for 30 minutes at 12 000 rpm. Part of the cell lysate was employed as the input. Another part of the cell lysate was incubated with 1 μg of anti‐Bcl‐2 antibody (Abcam Inc) or IgG (Abcam Inc) at 4℃ overnight. Afterwards, 10 μL of protein A agarose beads was washed 3 times with the cell lysis buffer. The pre‐treated protein A agarose beads were mixed with the antibody‐lysate mixture and incubated for a period of 2‐4 hours under 4°C conditions. The agarose beads were collected by means of centrifugation at 2664 g at 4°C for 3 minutes. The beads were then washed 3 times with 1 mL of cell lysis buffer. The concentration of the protein binding in the beads was determined and adjusted to an equal amount. The protein was mixed with an equal volume of 2× sodium dodecyl sulphate (SDS), and boiled for 5 minutes. The expression of Beclin1 was measured by Western blot analysis methods. The expression of Bcl‐2 binding to Beclin1 was investigated based on the aforementioned method, where the anti‐Beclin1 antibody (Abcam Inc) was utilized.
7
Western Blot Analysis of Protein Expression
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Western blot experiments were performed as previously reported[15 (link)]. Cells were lysed with RIPA buffer (YEASEN, China) containing PMSF (Beyotime, China). After determining the protein concentration using the BCA protein kit (Thermo Scientific, USA), the protein lysate was mixed with 5 X SDS loading buffer (EpiZyme, China) and heated at 100°C for 10 min. A total of 30 μg of proteins were separated and transferred to a PVDF membrane. Then, the membrane blocked by 5% nonfat milk for 1 h was incubated with an anti-ZNF268 antibody (1:1000; Abmart, China), anti-GAPDH antibody (1:1000; Abcam, USA), anti-BCL-2 antibody (1:1000; Abcam, USA) and anti-Bax antibody (1:1000; Abcam, USA) in 4°C overnight. And the membrane was dyed by ECL after the co-incubation of the secondary antibody (1:5000; Jackson immunoresearch, USA) for 1 h.
8
Western Blot Analysis of Brain Proteins
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Protein lysates from the various specialized structures of mouse brains or rat C6 glioma cells was run on a SDS-acrylamide gel electrophoresis (SDS-PAGE) in a vertical electrophoresis apparatus and transferred to a PVDF membrane in blotting buffer (20 mmol/L Tris-base, 150 mmol/L glycine and 20 % methanol) to make proteins accessible to the detection by monoclonal antibodies [23] . The commercially available monoclonal antibodies including anti-GAPDH antibody (Abcam, England), anti-Bcl2 antibody (Abcam, England), anti-DAPK1 antibody (Abcam, England), anti-TH antibody (Santa Cruz Biotechnology, USA) and anti-NMDAR1 antibody (Sigma, USA) were employed in this study. The signals of protein bands were detected using an enhanced chemiluminescent (ECL) system (GE Healthcare Bio-Sciences Corp.).
9
Protein Expression Analysis in Cancer Cells
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Following the protocols of the manufacturer, RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) was used for cellular protein extraction. The BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) was selected for the detection of protein concentrations. The primary antibodies included rabbit anti-Ki67 antibody (1:25, ab833, Abcam), anti-proliferating cell nuclear antigen (PCNA) (1:1,000, #13110, Cell Signaling), anti-Survivin (1:1,000, #2808, Cell Signaling), anti-Bax (1:1,000, #5023, Cell Signaling), anti-Bcl-2 antibody(1:500, ab196495, Abcam), anti-CD44 (1:100, #37259, Cell Signaling), anti-SOX2 (1:1,000, #14962, Cell Signaling), anti-OCT4 (1:1,000, #2890, Cell Signaling), anti-ALDH1 (1:1,000, #54135, Cell Signaling), β-catenin (1:1,000, #8480, Cell Signaling), c-Myc (1:1,000, #18583, Cell Signaling), and cyclin D1 (1:1,000, #2890, Cell Signaling). Goat anti-rabbit immunoglobin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated for 1 h at 37 °C. An automatic digital gel image analysis system, Bio-Rad CFX-96 (Bio-Rad, Hercules, CA, USA) was used to determine and analyze band density.
10
Western Blot Analysis of Apoptotic Proteins
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Cells were seeded, cultured and exposed with different treatments for 72 h. Whole cell protein lysates were prepared according to standard protocol using RIPA buffer (0.5 M Tris–HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA). Protein (50 μg) was loaded per well of a 12% SDS PAGE gel using electrophoresis buffer (0.192 M glycine, 25 mM Tris, 0.1% SDS). After electrophoresis, the gel was transferred onto a PVDF membrane (Bio-Rad Laboratories, Inc., CA, USA) using transfer buffer (0.192 M glycine, 25 mM Tris, 0.025% SDS, 10% methanol). Membranes were blocked in TBS-T with 5% non-fat dry milk and incubated overnight with the primary anti-Bax (1:1000, Abcam), anti-Bcl-2 antibody (1:250, Abcam) or anti-cleaved-caspase-3 (1:1000, Cell signaling), then incubated with secondary HRP-linked antibody (1:5000) (KPL Inc., USA). Detection was done by Abcam Optiblot ECL Detect Kit (Abcam, USA). Anti-β-tubulin antibody (1:20000) (Sigma-Aldrich Co., St. Louis, MO, USA) was used to for loading correction.
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