Cells were lysed in RIPA Lysis Buffer (MA0151, Dalian Meilun Biotechnology Co., Ltd., China) containing a protease inhibitor cocktails (FD1001, Fudebio, Hangzhou, China) on ice. Equal amounts of total protein from different samples were separated by SDS-PAGE gels at 100 V for 1.5 h and transferred onto 0.22 μm polyvinylidene difluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ) at 280 mA for 1.5 h. Then, the membranes were blocked with 5% skim milk powder in TBST for 1 h at room temperature and treated with specific primary antibodies overnight at 4 °C. The next day, the membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (FDM007 and FDR007, Fudebio, Hangzhou, China). Each band was detected using an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). Anti-GAPDH, anti-CREB3, anti-Bcl-2, anti-mmp2, anti-Vimentin and anti-E-cadherin antibodies were purchased from Abcam (Cambridge, UK). The anti-c-Jun, anti-cleaved-caspase 3 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); anti-mmp9 antibody was purchased from ABclonal (Boston, MA, USA); and anti-N-cadherin antibody was purchased from Proteintech (Chicago, USA). Primary Antibody Dilution Buffer was purchased from Dalian Meilun Biotechnology Co., Ltd. (MB9881, Dalian, China).
Ripa lysis buffer
Manufactured by Meilun
Sourced in China
RIPA lysis buffer is a detergent-based buffer used for cell lysis and protein extraction. It contains a mixture of ionic and non-ionic detergents, as well as other components to facilitate efficient cell lysis and protein solubilization. The buffer is suitable for use in a variety of applications, including Western blotting, immunoprecipitation, and other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Fd8030, by Fudebio (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Protease inhibitor cocktails, by Fudebio (2 mentions)
Bovine serum albumin (bsa), by Meilun (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab7090, by Abcam (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Anti β actin, by Immunoway (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Bca kit, by Meilun (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor cocktail, by Selleck Chemicals (2 mentions)
Primary antibody dilution buffer, by Meilun (1 mentions)
Fdm007, by Fudebio (1 mentions)
Fd1001, by Fudebio (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
30 protocols using ripa lysis buffer
1
Protein Expression Analysis by Western Blot
2
Protein Expression Analysis by Western Blot
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The cells were collected and RIPA lysis buffer (Meilun Biotechnology Co., Ltd., Suzhou, China) containing PMSF (1 mM) (Meilun Biotechnology Co., Ltd., Suzhou, China) was added for protein extraction. Total protein concentration of samples was detected by BCA protein assay. After diluting the protein sample to the same concentration with RIPA lysis solution, added 5 × loading buffer, boiled in 100 °C water bath for 5 min, and store at -80 °C after cooling on ice. 30 μg total protein samples were taken from each group, separated by SDS-PAGE gels electrophoresis and transferred to PVDF membranes. After sealing with 5% bovine serum albumin (Albumin from bovine serum, BSA), the membranes were incubated with primary antibodies anti-VEGF (1:2000, 66,828–1-IG; proteintech), anti-HIF-1α (1:2000, 66,730–1-IG; proteintech), or anti-GAPDH (1:2000, 60,004–1-IG; proteintech). Following incubation with the secondary antibody solution of HRP Goat Anti Mouse IgG (H + L) (1:5000, proteintech). The PVDF membranes were color-coded by chemiluminescence method and luminescence detection by gel imaging system Versa DocTM Imaging System to collect strip images. GAPHD was used as internal reference to calculate the relative expression levels of each target protein by Image Lab analysis software (National Institutes of Health).
3
Protein Expression Analysis Protocol
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Protein samples were prepared using RIPA lysis buffer (Meilun Biotech Co., Ltd.) containing protease inhibitors on ice for 15 min, and were then centrifuged at 12,000 × g for 15 min at 4°C. Protein concentrations were measured using the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein were separated by 10% SDS-PAGE, and transferred to PVDF membranes (MilliporeSigma). They were then incubated with TRIS-buffered saline (TBST) containing 5% skim milk at 37°C for 2 h, followed by incubation with primary antibodies targeting CDCA5 rabbit mAb (dilution, 1:2,000; cat no. A4044; ABclonal Science, Inc.), OCT4 rabbit mAb (dilution, 1:2,000; cat no. T55781; Abmart Inc.), c-MYC rabbit mAb (dilution, 1:2,000; cat no. T55150; Abmart Inc.), SOX2 rabbit mAb (dilution, 1:2,000; cat no. T55268; Abmart Inc.) and GAPDH rabbit mAb (dilution, 1:2,000; cat no. A19056; ABclonal Science, Inc.) overnight at 4°C. Following three 5-min washes in TBST, membranes were incubated with HRP goat anti-rabbit secondary antibodies (dilution, 1:2,000; cat no. AS014; ABclonal Science, Inc.) at room temperature for 1.5 h. Chemiluminescent signals were detected using enhanced chemiluminescence detection reagents (Meilun Biotech Co., Ltd.).
4
Comprehensive Protein Analysis Protocol
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Bicinchoninic acid (BCA) protein assay kit (#MA0082-1) and RIPA lysis buffer (#MA0153) were purchased from Meilunbio (Dalian, China). L-arginine (L-Arg) monohydrochloride (#A6969), Collagenase IV (#C9407), Disulfiram (#HY-B0240) and DAPI (#D9542) were from Sigma-Aldrich (St. Louis, MO, USA) of Merck. GSDMD antibody (#AF4012) was from Affinity (Shanghai, China), REDD1 (Regulated in Development and DNA Damage Responses 1). Antibody (#sc-376671) and p-NF-Kb (p-P65) (#sc-398442) were from Santa Cruz Biotechnology (CA, USA). Resveratrol (#A2122398) was from Aladdin (Shanghai, China). TXNIP(#ab188865) and HIF-1α (#ab1) were from Abcam (Boston, MA, USA). Malondialdehyde (MDA) assay kit (#S0131S), catalase (CAT) (#S0051) assay kit, total glutathione peroxidase (GPX) assay kit with NADPH (#S0058) and anti-β-actin antibody (AF0003) were purchased from Beyotime Biotech (Shanghai, China). Mouse IL-1β ELISA kit (E-EL-M0037c) was purchased from Elabscience Biotechnology Co. Ltd. (Wuhan, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) of Merck.
5
Isolation and Quantification of Tubulin Fractions
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The free and polymerized tubulin fractions were isolated using a method previously described by Li et al (Li et al. 2015 (link)). Cells (2 × 106) grown in 35-mm petri dish plates were washed twice with a microtubule stabilization buffer (MTSB, 0.1 M piperazine-N, N′-bis (PIPES), 2 mM ethylene glycol-bis (β-aminoethylether) N,N,N′,N′-tetraacetic acid (EGTA), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM MgCl2, and 2 M glycerol). The cells were incubated with MTSB + 0.1% Triton X-100 + protease inhibitor cocktail (1:200) for 30 min, and the supernatant was collected as the free tubulin fraction. The Triton X-100 insoluble fraction, corresponding to the polymerized tubulin, was then solubilized in a RIPA lysis buffer (Dalian Meilun Biology Technology Co., Ltd., China) with a protease inhibitor cocktail. Cells were lysed for 30 min and centrifuged at 15,000g for 15 min at 4 °C. The polymerized and free tubulin fractions were quantified using western blotting analysis.
6
Western Blot Analysis of EMT Markers
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NPC cells were lysed in RIPA lysis buffer (Meilun Biotechnology Co., Ltd., Dalian, China) containing protease inhibitor cocktails (Fudebio, Hangzhou, China). Then, total protein from different samples (30 μg/per lane) was separated by SDS-PAGE and transferred onto 0.22 μM PVDF membranes (Amersham Bioscience, Piscataway, NJ, United States). After that, the membranes were blocked with 5% skimmed milk in TBST for 1 h and incubated with specific primary antibody overnight at 4°C. After being washed with TBST, the membranes were incubated with the secondary antibody for 1 h at room temperature. Next, the protein bands were developed using an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). Antibodies were listed: anti-E-cadherin (Abcam, ab76319, 1:2,000), anti-Vimentin (Abcam, ab92547, 1:2,000), anti-PIK3R1 (Abcam, ab191606, 1:1,000), anti-ERBB2 (Abcam, ab237715, 1:1,000), anti-GAPDH (Abcam, ab8245, 1:3,000), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719, 1:5000), and goat anti-rabbit IgG H&L (HRP) (Abcam, ab205718, 1:5,000).
7
Protein Extraction and Western Blot Analysis
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The samples were lysed with RIPA lysis buffer (Meilunbio, China) for protein extraction. The supernatants were collected for protein analysis after the lysates were centrifuged at 14,000 × g for 15 min at 4°C. Protein samples were separated via 10% SDS-PAGE (Meilunbio, China) and transferred to PVDF membranes (0.2 μm, Millipore, Bedford, MA, USA). After probing with rabbit anti-LTF (1:500, BOSTER, China) and anti-β-actin (1:1000, Proteintech, China) overnight at 4°C, the membranes were incubated with goat anti-rabbit IgG (10000, Immunoway, USA) for 1 h. Protein bands were detected using an ECL kit (Meilunbio, China). Densitometry was performed with ImageJ software.
8
Immunoprecipitation and Western Blot Analysis
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The total protein was extracted using RIPA Lysis Buffer (Meilunbio, Dalian, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was chosen for the total protein separation, and the proteins were then transferred to nitrocellulose membranes (the membranes were cut horizontally). The membranes were incubated with primary antibodies, including anti-p62/SQSTM1 (Abcam (Cambridge, UK), Cat# ab207305), anti-LC3B (Abcam, Cat# ab192890), anti-ACTB (Sangon Biotech (Shanghai, China), No. D110001), anti-PARP (Proteintech (Rosemont, IL, USA), Cat No. 13371-1-AP), anti-cleaved caspase3 (Cell Signaling Technology (Danvers, MA, USA), 5A1E), anti-SIRT1 (Cell Signaling Technology (Danvers, MA, USA), C14H4), anti-Acetylated lysine (Cell Signaling Technology (Danvers, MA, USA), #9441), and anti-Beclin1 (Proteintech, Cat No. 11306-1-AP). Immunoprecipitation was carried out either by incubating Anti-Flag beads (MCE (Addison, IL, USA), Cat. No. HY-K0207) at 4 °C with lysate overnight or by incubating an appropriate antibody with cell lysate for 4–6 h, followed by incubating Protein A/G immunoprecipitation beads overnight. Immunoprecipitates were washed three times with cold lysis buffer and eluted with SDS loading buffer by boiling for 10 min. The original western blot figures can be found in Figure S1 .
9
Preparation of Fluorescent Nanomaterials
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RIPA lysis buffer, BSA, EDTA, Tris-HCl, protease inhibitor cocktail, Coomassie blue, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) and DiI were purchased from Dalian Meilun Biotechnology. Lecithin was purchased from Solarbio, cholesterol was purchased from Shanghai Yuanye BioTechnology, and 2-distearoyl-sn-glycero-3-phos-phoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was purchased from AVT. Ferredoxin and FNR from Spinacia oleracea were purchased from Sigma-Aldrich. All other chemicals were purchased from Sigma-Aldrich unless specifically mentioned.
10
MUC-1 Protein Expression Analysis
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IEC-6 cells were lysed by RIPA lysis buffer (Meilunbio, Dalian, Liaoning, China). SDS loading buffer was added to the sample and incubated at 100°C for 10 min. Sample proteins were electrophoretically separated by 30% SDS-PAGE gels (Servicebio) and transferred to a 0.45µm polyvinylidene difluoride (PVDF) membrane. The proteins were blocked with 5% skim milk in Tris-buffered saline. Then samples were incubated with primary anti-MUC-1 (Servicebio) and anti-β-actin (Abclonal, Wuhan, Hubei, China) at 4°C overnight. Next, the samples were incubated with HRP-labeled goat anti-rabbit IgG H&L (Servicebio). Sample bands were visualized using enhanced chemiluminescence (ECL) and analyzed with the ProteinSimple instrument (Bio-Techne, Minnesota, USA).
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