Tissues were lysed using RIPA buffer (Beyotime, Shanghai, China), and lysates were loaded onto 12% bis–tris-acrylamide gels (Thermo Fisher, Waltham, MA, USA). The separated protein bands were electrotransferred onto polyvinylidene fluoride membranes (Millipore, Bradford, MA, USA), and then immersed in 5% nonfat dry milk (Solarbio, Beijing, China). After that, the membranes were incubated with anti-BCL2-associated x protein (anti-Bax) (1:5000; Abcam, Cambridge, UK), anti-matrix metalloprotein 2 (anti-MMP2) (1:5000; Abcam, Cambridge, UK), anti-MMP9 (1:3000; Abcam, Cambridge, UK), anti-caspase 3 (anti-t-caspase 3) (1:5000; Abcam, Cambridge, UK), anti-cleaved-caspase 3 (anti-C-caspase 3) (1:8000; Abcam, Cambridge, UK), anti-proliferating cell nuclear antigen (anti-PCNA) (1:3000; Abcam, Cambridge, UK), anti-MYC proto-oncogene, bHLH transcription factor (anti-c-Myc; 1:1000; Abcam, Cambridge, UK), anti-KRAS proto-oncogene, GTPase (anti-K-RAS; 1:1000; Abcam, Cambridge, UK), anti-B-Raf proto-oncogene, serine/threonine kinase (anti-BRAF; 1:2000; Abcam, Cambridge, UK) and anti-GAPDH (1:15,000; Abcam, Cambridge, UK), respectively. Then, the membranes were incubated with secondary antibody (1:8000; Abcam, Cambridge, UK). Finally, RapidStep ECL Reagent (Millipore, Bradford, MA, USA) was used to visualize the protein bands. GAPDH was chosen as a control.
Nonfat dry milk
Manufactured by Solarbio
Sourced in China
5% nonfat dry milk is a laboratory reagent used as a blocking agent in western blotting, enzyme-linked immunosorbent assays (ELISA), and other immunodetection techniques. It helps to reduce non-specific binding of antibodies and proteins during the blocking and incubation steps of these assays.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Solarbio (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti braf, by Abcam (1 mentions)
Anti c caspase3, by Abcam (1 mentions)
Anti kras, by Abcam (1 mentions)
Rapidstep ecl reagent, by Merck Group (1 mentions)
Bis tris acrylamide gel, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti c myc, by Abcam (1 mentions)
Anti mmp2, by Abcam (1 mentions)
Anti mmp9, by Abcam (1 mentions)
Anti pcna, by Abcam (1 mentions)
Ecl reagent, by Affinity Biosciences (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Chemidoctm xrs imaging system, by Bio-Rad (1 mentions)
Chemiluminescence reagent, by Merck Group (1 mentions)
β actin, by Media Cybernetics (1 mentions)
6 protocols using nonfat dry milk
1
Western Blot Analysis of Apoptosis and Proliferation Markers
2
Western Blot Analysis of Inflammatory Markers
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Total protein was extracted with RIPA (radio immunoprecipitation assay) lysate. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) 50 μg of protein samples were subjected to SDS-PAGE and transferred onto PVDF membranes (Solarbio, Beijing, China). The membrane was blocked with 5% nonfat dry milk (Solarbio, Beijing, China) for 1.5 h, then incubated with specific primary antibodies at 4 °C overnight. Following incubation with HRP-conjugated secondary anti-rabbit antibody (7074, Cell Signaling Technology, Danvers, MA, USA), membranes were examined with ECL reagent (KF005, Affinity Biosciences, Melbourne, Australia) and visualized on ChemiDocTM XRS + Imaging System (BioRad, Hercules, CA, USA). ImageJ software (U.S. National Institute of Health, Bethesda, MD, USA), was applied to detect and analyze the gray values of protein bands on the membrane. The following primary antibodies: transforming growth factor (TGF)-β, interleukin (IL)-10, tumor necrosis factor (TNF)-α, interleukin(IL)-6, NADPH oxidase 2 (NOX2), P22, arginase-1 (Arg-1), C–C motif chemokine ligand 3 (CCL3), interleukin(IL)-1β, monocyte chemoattractant protein-1 (MCP-1), C–X3–C motif chemokine ligand 1 (CX3CL1), and β-actin. All the above antibodies purchased form Proteintech, Beijing, China.
3
Western Blot Analysis of Autophagy Markers
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The cell samples were lysed with the tissue and cell lysate (Solarbio, Beijing, China) containing protease inhibitor after washing with ice-cold PBS. The cell lysates containing 15 μg of protein per sample were loaded into sodium-dodecyl sulfate polyacrylamide (SDS-PA) (Solarbio, Beijing, China) gels and separated by electrophoresis. After transferring the proteins onto a polyvinylidene fluoride (PVDF) membrane, the latter were blocked nonspecifically with 5% nonfat dry milk (Solarbio, Beijing, China) for 1 h at room temperature. The membranes were incubated overnight with the primary antibodies anti-LC3B (1:2000, Abcam, Cambridge, MA, USA), anti-p62/SQSTM1 (1:4000, Novus Biologicals, Littleton, NH, USA), and anti-β-actin (1:1000, Abcam, Cambridge, MA, USA) at 4 °C, and the blots were washed with TBST (Solarbio, Beijing, China) prior to incubating them with secondary antibody (1:2000, Abcam, Cambridge, MA, USA) at 37 °C for 1 h. Subsequently, the membranes were developed by exposure to chemiluminescence reagents (Millipore, Billerica, MA, USA) and visualized with ChemiDocTM Imaging Systems (Tanon-5200, Shanghai, China). The band density was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) for each group and normalized with β-actin.
4
Western Blot Detection of His-Tagged Proteins
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The proteins in polyacrylamide gel were blotted on nitrocellulose membrane using iBlot 2 Gel Transfer Device (Life Technologies). The nitrocellulose was blocked with 5% nonfat dry milk (Solarbio) in Tween 20 supplemented with tris (TBST) buffer for 30 min and washed with TBST buffer two times. The membrane was incubated in primary anti-His antibody (TransGen Biotech) for 2 hours and washed with TBST buffer two times, followed by incubation with second anti–horseradish peroxidase antibody (TransGen Biotech) for 1 hour and washed with TBST buffer three times. The resultant membrane was incubated with Clarity Western ECL Substrate (Bio-Rad) solution in a dark environment at room temperature for 1 min and imaged through the ChemiDoc MP system (Bio-Rad).
5
Protein Extraction and Western Blotting
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Total proteins were separately extracted from MIA PaCa-2 and Panc03.27 cells, which were treated as described in the transwell migration assay (a pre-experiment revealed irisin-induced AMPK phosphorylation persists for at least 24 h in MIA PaCa-2 cells, unpublished data). The cells were washed with pre-cooled 1× PBS and solubilized. After incubation in ice for 30 min, the lysates were centrifuged at 12000 rpm for 20 min at 4 °C. Protein concentration in the supernatant was measured using BCA Protein Assay Kit. The same amount of protein was loaded into each well of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to PVDF membranes (Millipore, CA, USA). The PVDF membranes were blocked with 5% nonfat dry milk (Solarbio, Beijing, China) in TBST (10 mM Tris-HCl, 150 mM NaCl, and 2% Tween) for 1 h at room temperature and incubated with primary antibodies (1:1000) at 4 °C overnight. Subsequently, PVDF membranes were washed with TBST thrice for 5 min and incubated with (HRP)-conjugated secondary antibodies for 1 h. Subsequently, the PVDF membranes were washed with TBST. Then the blots were visualized using an enhanced chemiluminescence substrate and detected using the Tanon 2500 chemiluminescence imaging system (Shanghai, China).
6
Amuc_1434 Protein Expression Analysis
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LS174T cells were treated with different concentrations of Amuc_1434*. Total proteins were separately extracted from LS174T cells and the total protein concentration was measured using a BCA Protein Assay Kit (C503051, Sangon Biotech Co. Ltd., Shanghai, China). The same amount of protein was loaded into each well for 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, CA, USA). The PVDF membranes were blocked with 10% nonfat dry milk (Solarbio, Beijing, China) in TBST (10 mM Tris-HCl, 150 mM NaCl and 2% Tween-20) at room temperature for 3 h, and incubated with primary antibodies (dilution ratio 1:1000, using PBS) at 4 °C overnight. Subsequently, PVDF membranes were washed three times with TBST for 15 min and incubated with HRP-conjugated secondary antibodies for 1 h. Subsequently, the PVDF membranes were washed three times for 15 min with TBST. The protein was detected with a super-sensitive electrochemiluminescence (ECL) luminescence reagent (MA0186, Meilunbio, Dalian, China).
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