AGS and MKN-28 cells were lysed with radioimmunoprecipitation assay lysis buffer containing protease inhibitor, the supernatant was collected after high speed centrifugation and heated in a water bath pot at 100 °C for 10 minutes to denaturalize the protein. The content of the protein was determined by bicinchoninic acid method. After that, Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmembrane were performed. Cells were incubated with primary antibodies at 4 °C overnight and then with the goat antirabbit secondary antibody (Wuhan Service Biotechnology Co, Ltd, GB23303, 1:2000) at room temperature for 1 hour. The primary antibodies include ZNF521 antibody (Abcam, ab189956, 1:1000), Bcl-2 antibody (Abcam, ab185002, 1:1000), BAX antibody (Abcam, ab32503, 1:1000), E-cadherin antibody (Abcam, ab194982, 1:1000), and N-cadherin antibody (Abcam, ab202030, 1:1000). Ultimately, color rendering was performed using hypersensitive enhanced chemiluminescence (ECL) (Hubei Biossci Biotechnology Co, Ltd.).
Ab185002
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab185002 is a monoclonal antibody that can be used for immunohistochemistry applications. It targets the CD4 antigen.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (26 mentions)
Ab181602, by Abcam (14 mentions)
Pvdf membrane, by Merck Group (13 mentions)
Ab205718, by Abcam (8 mentions)
Ab134175, by Abcam (5 mentions)
Ab32042, by Abcam (5 mentions)
Ab2302, by Abcam (4 mentions)
Ab179467, by Abcam (3 mentions)
Ab92547, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab13847, by Abcam (3 mentions)
Ab16663, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Ab6721, by Abcam (3 mentions)
Ab1416, by Abcam (3 mentions)
Ab53154, by Abcam (2 mentions)
Ab32147, by Abcam (2 mentions)
Ab62352, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Aprotinin, by Merck Group (2 mentions)
35 protocols using ab185002
1
Western Blot Analysis of Protein Expression
2
Phillyrin Modulates Oxidative Stress and Apoptosis in ARPE-19 Cells
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Human ARPE-19 cell line was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). Phillyrin (batch number Z17A8X34077, purity > 98%) was a product of the China Food and Drug Administration (Beijing, China). Dulbecco's Modified Eagle's Medium/nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin, and streptomycin solutions were purchased from Corning (NY, USA). Trypsin (0.05%) and phosphate buffered saline (PBS) were produced by Gibco, Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT), ML385, N-acetylcysteine (NAC)m and H2O2 solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Fas (ab82419), cytochrome c (ab90529), cleaved caspase-3 (ab2302), cleaved caspase-9 (ab2324), NQO1 (ab80588), Keap1 (ab118285), Bcl-2 (ab185002), Nrf2 (ab62352), CDK2 (ab32147), cyclin A (ab33911), cyclin E (ab181591), Bax (ab53154), β-actin (ab8226), p21 (ab188224), Histone H3 (ab1791), COX IV (ab16056) p53 (ab241556), pro caspase-3, pro caspase-8, and pro caspase-9 were obtained from Abcam, Cambridge, (MA, USA). Antibodies against p-p53 (9286S), cleaved caspase-8 (9496S), cleaved PARP (5625S), and HO-1 (86806S) were purchased from Cell Signaling Technology, Beverly, (MA, USA).
3
Western Blot Analysis of Apoptosis Regulators
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RIPA buffer (Biossci, Wuhan, China) was added to the cells in each group. After fully mixed, the lysis was incubated on ice for 30 min. Then, the lysis was centrifuged for 15 min at 12,000 r/min at 4°C, and the supernatant was collected. After SDS-PAGE, the proteins were transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA) and afterwards the membranes were blocked with 5% skimmed milk. Then, the membranes were incubated at 4°C overnight with primary antibodies anti-Bcl-2, Bax, MMP11 and β-actin. After washing the membranes with TBST, they were incubated with HRP-labeled secondary antibodies for 1 h at room temperature. Subsequently, equivoluminal liquid A and liquid B of the hypersensitive ECL chemiluminescence kit (Millipore, Bedford, MA, USA) was mixed and cover the PVDF membranes to react with antigen-antibody complex. Then, the protein bands were scanned. The antibodies used in this study included anti-MMP11 antibody (ab53143, 1:2000), anti-Bcl-2 antibody (ab185002, 1:2000), anti-Bax antibody (ab32503, 1:2000) and anti-β-actin antibody (ab179467, 1:2000), which were all available from Abcam (Shanghai, China).
4
Western Blot Analysis of Autophagy and Apoptosis Markers
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GC tissues and cells were lysed in radioimmunoprecipitation assay buffer (Sangon). Next, 35 μg of proteins were loaded on 10% Bolt™ Bis-Tris precast polyacrylamide gels (Invitrogen) to perform electrophoresis for 120 min. Then the separated proteins on the gels were moved onto polyvinylidene fluoride membranes (Millipore) through an electrotransfer apparatus, and then the membranes were immersed in 5% skim milk (Sangon) to block the binding of nonspecific proteins. After incubation with primary antibodies for 4 h and secondary antibodies for 1 h at room temperature, the combined specific protein complex was detected using ECL substrate kit (Abcam, Cambridge, UK) and target protein levels were analyzed by the ImageLab software version 4.1 (Bio-Rad, Hercules, CA, USA). Our antibodies were all purchased from Abcam: anti-B-cell lymphoma-2 (anti-Bcl-2; ab185002, 1: 1000), Bcl-2-associated X (anti-Bax; ab32503, 1: 1000), anti-light chain 3B (anti-LC3B; ab51520, 1: 1000), anti-p62 (ab109012, 1: 1000), anti-Beclin-1 (ab62557, 1: 1000), anti-KAT6B (ab58823, 1: 1000), anti-GAPDH (ab9485, 1: 3000), and anti-rabbit immunoglobulin G/horseradish peroxidase secondary antibody (ab205718, 1: 5000).
5
Cell Lysis and Protein Immunoblotting
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Cells were lysed with a buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaC1, 10% glycerol, 1.5 mM MgCl2, 5 mM EGTA, protease inhibitors (4 mM phenyl methylsulfonyl fluoride and 100 mg/ml aprotinin, Sigma-Aldrich), and phosphatase inhibitors (10 mM sodium orthovanadate and 20 mM sodium pyrophosphate, Sigma-Aldrich) and processed. For direct immunoblot analysis, we employed 15–30 μg of total cellular proteins, which were resuspended with 25 μl of loading buffer, boiled for 5 min, and loaded on SDS-PAGE for Western blot (WB). The antibodies for WB were used at the condition suggested by the suppliers: rabbit anti-NOTCH-1 (ab27526, 1/500, Abcam, UK), rabbit anti-Bcl2 (Ab185002, 1/500, Abcam), rabbit anti-human NUMB (ab-14140, 1/1000, Abcam), mouse anti-p53 (ab1101, 1/1000, Abcam), mouse anti-Myc (sc-40, 1/200, Santa Cruz Biotechnology, Texas, USA), and mouse anti-beta-actin (sc-81178, 1/1000, Santa Cruz Biotechnology). The WBs were acquired with the ChemiDoc MP Imaging System (Bio-Rad Laboratories Inc., California, USA), and the corresponding bands were quantified with Image Lab 6.1.0 (Bio-Rad Laboratories, Inc.). The p-value for the relative amount was obtained with the Student’s t-test.
6
Western Blot Analysis of Melanoma Proteins
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Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Sigma-Aldrich) was utilized for extracting total proteins from melanoma tissues and cells. At first, proteins were segregated on 10% sodium dodecyl sulfate-polyacrylamide gel (Sigma-Aldrich) and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich). After the membranes were blocked by 5% skim milk (Thermo Fisher Scientific), primary antibodies were incubated to the membranes overnight at 4°C. The primary antibodies used were as below: anti-E-cadherin (1:1000; ab40772, Abcam, Cambridge, United Kingdom), anti-N-cadherin (1:1000; ab76011, Abcam), anti-Vimentin (1:1000; ab92547, Abcam), anti-ITGA9 (1:1000; ab140599, Abcam), anti-proliferating cell nuclear antigen (anti-PCNA; 1:1000, ab92552, Abcam), anti-cyclinD1 (1;1000, ab134175, Abcam), anti-B-cell lymphoma-2 (anti-Bcl-2; 1:1000, ab185002, Abcam), anti-Bcl-2-associated X (anti-Bax; 1;1000, ab32503, Abcam) and anti-GAPDH (1:3000; ab181602, Abcam). At the room temperature, the incubation with goat anti-rabbit secondary antibody (1:5000; ab205718, Abcam) was up to 1 h. Eventually, the chromogenic reaction was carried out by the enhanced chemiluminescence (ECL) reagent (Sigma-Aldrich), and the protein bands could be observed using the Image J software (NIH, Bethesda, MD, USA).
7
Protein Expression Analysis in Clinical Samples
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The RIPA lysis buffer (Beyotime, China) was used extract total proteins from clinical tissues and cell lines. The proteins were separated by 10% sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the targeted protein bands were transferred onto PVDF membrane (Sigma, USA). Subsequently, the PVDF membranes were probed with the primary antibodies including anti-β-actin (#ab8226, Abcam, USA), anti-SOD2 (#ab13534, Abcam, USA), anti-NLRP3 (#ab214185, Abcam, USA), anti-CyclinD1 (#ab16663, Abcam, USA), anti-Cyclin E2 (#ab32103, Abcam, USA), anti-CDK2 (#ab32147, Abcam, USA), anti-CDK4 (#ab108357, Abcam, USA), anti-CDK6 (#ab124821, Abcam, USA), anti-Caspase 3 (#ab13847, Abcam, USA), anti-Bax (#ab32503, Abcam, USA), anti-Bcl-2 (#ab185002, Abcam, USA), anti-Caspase 1 (#ab238979, Abcam, USA) and anti-IL-1β (#ab33774, Abcam, USA). After that, the membranes were incubated with anti-Rabbit IgG antibody at 4°C for 24h, and the enhanced chemiluminescent (ECL) system was employed to detect the protein bands. Finally, all the protein bands were quantified by Image J software. The primers sequences of the target genes were listed in Table 1 .
8
Quantifying Apoptosis Regulatory Proteins
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Western blot analysis was made to assess proteins in transfected cells in accordance with a previous study [33 (link)]. RIPA lysis buffer with a protease inhibitor cocktail was prepared to lyse cells. Next, total protein was separated by SDS PAGE and transferred onto PVDF (polyvinylidene difluoride) membranes. After being blocked with 5% non-fat milk, the membrane was incubated with primary antibodies: anti-Bax (abcam, ab32503), anti-Bcl-2 (abcam, ab185002), anti-YY1 (abcam, ab109237), and anti-GAPDH (abcam, ab8245) at 4 °C overnight. After incubation with specific antibodies, the blots were incubated with the secondary antibody and visualized with enhanced chemiluminescence. Then, GAPDH was used as an internal control. All experimental procedures were repeated for three times.
9
Apoptosis and Metastasis Pathways Analysis
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Harvested cells were lysed with RIPA buffer (Cat. No. 89901; Thermo Fisher Scientific Co. Ltd., Waltham, MA, USA). Proteins were quantified using BCA kits (Cat. No. P0012S; Beyotime Biotechnology, Shanghai, China), resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Non-specific binding was blocked with 5% skim milk in PBS at room temperature for 1 h. Membranes were immersed in primary antibodies to Caspase 3, Caspase 9, Bax, Bcl-2, MMP-2, MMP-9, and β-actin (Cat. No. ab13847, ab32539, ab32503, ab185002, ab92536, ab76003, ab8226 respectively; Abcam Plc., Cambridge, UK) diluted 1:1000, and left at 4°C overnight. Membranes were washed, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Cat. No. ab205718; Abcam Plc. diluted 1:1000) for 1 h at 37°C, then washed with PBS. Proteins were visualized using ECL luminescent reagent (Cat. No. 34096; Thermo Fisher Scientific Co., Ltd.). The internal reference protein was β-actin; the relative expression level of the test protein was calculated as the gray value of the test band/gray value of β-actin.
10
Extracting and Analyzing Cellular Proteins
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Total proteins were extracted using Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China), and the proteins were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Then, the membranes were placed in skim milk for blockage and probed with the primary antibodies against COMMD8 (K12416; 1:500; bjbalb, Beijing, China), Cyclin D1 (ab16663; 1:200; Abcam, Cambridge, MA. USA), Ki67 (ab16667; 1:1000; Abcam), B cell lymphoma/lewkmia-2 (Bcl-2) (ab185002; 1:1000; Abcam), Bcl-2 associated X Protein (Bax) (ab32503; 1:1000; Abcam), lactate dehydrogenase A (LDHA) (ab125683; 1:1000; Abcam), CD63 (ab134045; 1:1000; Abcam), CD81 (ab109201; 1:1000; Abcam) and β-actin (ab8227; 1:2000; Abcam) at 4°C for overnight. After the probe of the goat-anti-rabbit secondary antibodies on the next day, the protein signals were emerged using the enhanced chemiluminescent reagent (Beyotime) under an imaging system (Bio-Rad).
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