Cells were lysed and the proteins were separated in 10% SDS-PAGE, then transferred to a polyvinylidene difluoride membrane. After blocked with 5% non-fat milk the membranes were incubated with the following primary antibodies at 4°C overnight. Anti- Siva 1 antibody (sc-48767, Santa Cruz Biotechnology), anti- cleaved caspase-3 polyclonal antibody (ab2302, Abcam, Cambridge, UK), anti-Bax antibody (BA0315, Boster, Hubei, Wuhan, China), anti-Bcl-2 polyclonal antibody (BA0412, Boster, Hubei, Wuhan, China) were used. After washing with TBST 4 times for 5 min, the membranes were incubated with anti-rabbit or anti-mouse or anti-goat HRP-conjugated secondary antibody. The protein bands were visualized with a ECL reagent (Wanleibio Co., Ltd.) by the ImageQuant LAS 4000 system (GE Healthcare Life Sciences, Logan, UT, USA). β-Actin (A5316, Sigma) was used for equal loading.
Ba0412
Manufactured by Boster Bio
Sourced in China
BA0412 is a lab equipment product. It serves as a core function in laboratory settings, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ba0315, by Boster Bio (3 mentions)
Ba0315 2, by Boster Bio (3 mentions)
Ab2302, by Abcam (2 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Anti bcl 2, by Boster Bio (1 mentions)
β actin, by Merck Group (1 mentions)
Anti bax antibody, by Boster Bio (1 mentions)
Las 4000, by Cytiva (1 mentions)
Ab21685, by Abcam (1 mentions)
Af0096, by Affinity Biosciences (1 mentions)
Af3247, by Affinity Biosciences (1 mentions)
Microscope imaging system, by Olympus (1 mentions)
Ab182422, by Abcam (1 mentions)
Ab178846, by Merck Group (1 mentions)
Sc 271430, by Abcam (1 mentions)
Med 6.0 cmias image analysis system, by Motic (1 mentions)
Anti igf2bp3 14642 1 ap, by Proteintech (1 mentions)
Hrp 60004, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
8 protocols using ba0412
1
Western blot analysis of apoptosis-related proteins
2
Aortic Protein Expression Analysis
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Total protein was extracted from the frozen thoracic aorta of rats and HUVECs with RIPA lysis buffer containing protease inhibitor cocktail. The protein concentrations were determined by the Bradford protein assay kit. Equal amounts of protein lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes followed by a block with 5% skimmed milk at 4°C for 1.5 h. Subsequently, the membranes were incubated with primary antibodies against eNOS (AF0096, Affinity, 1:1,000) and p-eNOS (AF3247,Affinity, 1:1,000), c-jun N-terminal kinase (JNK BS1544, Bioworld, 1:500) and p-JNK (BS4322, Bioworld, 1:500), B-cell lymphoma-2 (Bcl-2, BA0412, Boster, 1:800), BCL2-Associated X (Bax, BA0315-2, Boster, 1:800), inositol-requiring enzyme 1α (IRE1α, bs-8680R, Bioss, 1:1,000), glucose regulated protein 78 (GRP78, ab21685, Abcam, 1:1,000), CHOP, respectively. β-actin (M02014-5, Boster, 1:1,500) was used as the internal reference. Antibody binding was detected with enhanced chemiluminescent agent and quantified with Image J software. Each experiment was repeated three times.
3
Immunohistochemical Analysis of Human GBM and Mouse CDA
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The immunohistochemical tissues of human and mouse were obtained from patients of the Department of Neurosurgery, Tongji Hospital, Huazhong University of Science and Technology (Wuhan, China) and mouse CDA model. Immunohistochemistry (IHC) primary antibody used in this study includes Goat anti-LRIG3 (1:50, R&D, AF3495), Rabbit anti-CD163 (1:100, Abcam, ab182422), Rabbit anti-IBA1 (1:2000, Abcam, ab178846), Rabbit anti-NETO2 (1:200, Sigma-Aldrich, HPA013180), Mouse anti-ARG1 (1:200, Santa Cruz, sc-271430), Rabbit anti-CD8 alpha (1:500, Abcam, ab209775) Rabbit anti-BAX (1:300, Boster, BA0412), and Rabbit anti-BCL2 (1:300, Boster, BA0315-1). The IOD scores of LRIG3, CD163, IBA1, ARG1 or CD8A in human GBM or mouse CDA models were quantified by software imageJ under ×400 magnified microscope with an Olympus Imaging System Microscope in the whole regions of each tumor specimen.
4
Immunohistochemical Analysis of Apoptosis Markers
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Immunohistochemical staining was performed using a commercial kit, according to the manufacturer’s instructions (ZSGB-BIO, Beijing, China). Primary antibodies used in this study were anti-Apaf-1 (1:200, BA2373), anti-Bax (1:300, BA0315), anti-Bcl-2 (1:200, BA0412) and anti-active caspase-3 (1:300, BA3968), anti-active caspase-9 (1:300, BA0690), all obtained from Boster, Co., Ltd., Beijing, China.
Apaf-1, Bax, Bcl-2, and active caspase-3, active caspase-9 positive signals were observed as brown or yellow granular masses in cells, and their intensities were measured using Motic Med 6.0 CMIAS Image Analysis System (Motic China Group, Co., Ltd., China). A total of 15 fields per gerbil (three fields per section, five sections per gerbil, 400× magnification) were randomly selected and analyzed. The positive staining intensity was calculated as the ratio of the stained area to the total field assessed (Du et al., 2015 (link)).
Apaf-1, Bax, Bcl-2, and active caspase-3, active caspase-9 positive signals were observed as brown or yellow granular masses in cells, and their intensities were measured using Motic Med 6.0 CMIAS Image Analysis System (Motic China Group, Co., Ltd., China). A total of 15 fields per gerbil (three fields per section, five sections per gerbil, 400× magnification) were randomly selected and analyzed. The positive staining intensity was calculated as the ratio of the stained area to the total field assessed (Du et al., 2015 (link)).
5
Protein Expression Analysis in Cultured Cells
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Total protein was extracted from cultured cells with a Protein Extraction Kit (Kaiji, Nanjing, China). Proteins were separated on SDS–PAGE gels and transferred onto PVDF membranes (0.45-μM pore, Millipore). Blots were probed with anti-IGF2BP3 (14642-1-AP, Proteintech), anti-RCC2 (16755-1-AP, Proteintech), anti-BCL2 (BA0412, Boster Bio), anti-BAX (BA0315-2, Boster Bio), anti-cleaved Caspase3 (29034, Signalway Antibody), and anti-GAPDH (HRP-60004, Proteintech) antibodies. Anti-rabbit or anti-mouse HRP-conjugated secondary antibodies were used prior to ECL detection.
6
Western Blot Analysis of Apoptosis-Related Proteins
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RIPA Lysis Buffer (Beyotime) was used to extract total protein, and BCA Protein Assay Kit (Beyotime) was used to quantify the protein. Afterwards, protein samples (30 µg) were separated by 10% SDS-PAGE gel and transferred to PVDF membranes (Beyotime). Next, the membranes were blocked with skimmed milk for 2 h. After incubating with primary antibodies against Bcl-2 (26Kda, 1:2,000, BA0412, Boster, Wuhan, China), Bax (20Kda, 1:1,500, BA0315-2, Boster), Cleaved-caspase 3 (17Kda, 1:1,000, AC033, Beyotime), FOXO1 (82Kda, 1:2,000, AF603, Beyotime), or GAPDH (36Kda, 1:2,000, A00227, Boster), the membrane was then incubated with HRP Conjugated AffiniPure Goat Anti-rabbit/mouse IgG (H + L) (1:10,000, BA1056, Boster). The protein signals were visualized using BeyoECL Star (Beyotime). EasySee Western Marker (25-90Kda, DM201-01, Transgen Biotech, Beijing, China) was used as a molecular weight standard.
7
Retinal Protein Expression Profiling
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The retinal tissues were lysed on the ice, followed by protein extraction. The protein concentration was determined by BCA assay kit (Wanleibio, Shenyang, China). The proteins were subjected to SDS-PAGE (8–13%) and transferred to PVDF membranes. Then, membranes were blocked with non-fat milk dissolved in Tween-20/TBS buffer. Subsequently, the membranes were incubated with primary antibodies against NgR (1:10,000; ab184556; Abcam, Cambridge, UK), RhoA (1:200; sc-197; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Rock1 (1:200; sc-374388; Santa Cruz Biotechnology, Inc.), Bcl-2 (1:400; BA0412; Boster, Wuhan, China), Bax (1:400; BA0315; Boster), Caspase-3 (1:1000; ab2302; Abcam), F-actin (1:500; ab205; Abcam), growth-associated protein-43 (GAP-43) (1:200; sc-33705; Santa Cruz Biotechnology, Inc.) at 4°C overnight and then with secondary antibody (1:5,000; horseradish peroxidase-labeled IgG; WLA023 and WLA024; Wanleibio) for 45 min at 37°C. The protein bands were visualized via ECL reagent and analyzed by Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).
8
Western Blot Analysis of Bcl-2 and β-Actin
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Cell samples were lysed with 50 mM Tris-base, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate (Sigma-Aldrich, D6750-25G), and 1 mM phenyl methane sulfonyl fluoride (Sigma-Aldrich, 78830-5G). Proteins (20 to 60 μg) were size-fractionated by 12% SDS-PAGE, and then transferred to PVDF (polyvinyl difluoride) membranes (Millipore, ISEQ00010). After blocking, the membranes were incubated with primary antibodies: β-actin (1:1000, sc-69879, Santa Cruz), Bcl-2 (1:400, BA0412, Boster). After washing with Tris-buffered saline containing 0.1% Tween 20 (TBST; Beyotime, ST825), the blots were probed with secondary antibodies (ZB-2305 and ZB-2301, ZSGB-Bio, Beijing, China) for 1 h at room temperature. Target proteins were examined using enhanced chemiluminescence reagents (Millipore, WBKLS0500). Band intensity was determined by densitometry using Image J software. The relative intensity of each band was standardized to the corresponding internal control. For relative quantification, the intensity of each band was standardized to the corresponding internal control and normalized to the intensity of the band of control group.
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