Twenty micrograms of protein sample was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). After blocking in 10% milk in Tris-buffered saline/Tween-20 (TBST) for 2 h at room temperature, the nitrocellulose membranes were incubated with the following primary antibodies overnight at 4°C: anti-LC3 monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA, 1: 1000), anti-P62 monoclonal antibody (Abcam, Cambridge, UK, 1:10000), anti-β-actin monoclonal antibody (Bioss, Beijing, China, 1:1000), and anti-DERL1 polyclonal antibody (Abcam, Cambridge, UK, 1:2000). The membranes were then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (BOSTER, Wuhan, China, 1:10000) for 2 h at room temperature, followed by expression detection with an electrochemiluminescence (ECL) system (Gene Gnome 5, Synoptics Ltd., UK).
Nitrocellulose membrane
Manufactured by Merck Group
Sourced in United States, Germany, China, United Kingdom, Ireland, France, Morocco, Italy, Australia, Sao Tome and Principe, Canada, Macao, Spain, India
Nitrocellulose membranes are a type of laboratory equipment used for various applications, such as protein blotting, DNA/RNA transfer, and immunodetection. They are made from purified cellulose that has been chemically treated to create a porous structure, allowing efficient binding and transfer of biomolecules. These membranes provide a reliable and versatile platform for a range of research and analytical procedures.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (114 mentions)
Ripa lysis buffer, by Beyotime (113 mentions)
Protease inhibitor cocktail, by Roche (112 mentions)
Ripa buffer, by Beyotime (96 mentions)
Bca protein assay kit, by Beyotime (86 mentions)
Protease inhibitor cocktail, by Merck Group (72 mentions)
Quantity one software, by Bio-Rad (67 mentions)
Odyssey infrared imaging system, by LI COR (66 mentions)
Gapdh, by Cell Signaling Technology (64 mentions)
β actin, by Merck Group (55 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (53 mentions)
β actin, by Santa Cruz Biotechnology (48 mentions)
β actin, by Cell Signaling Technology (47 mentions)
Bovine serum albumin (bsa), by Merck Group (37 mentions)
Bca kit, by Thermo Fisher Scientific (37 mentions)
β actin, by Abcam (35 mentions)
Ripa buffer, by Merck Group (35 mentions)
Ripa buffer, by Thermo Fisher Scientific (33 mentions)
Gapdh, by Abcam (32 mentions)
Odyssey imaging system, by LI COR (32 mentions)
2 825 protocols using nitrocellulose membrane
1
Western Blot Analysis of Autophagy Markers
2
Immunoblotting Assay for Protein Expression
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The nitrocellulose membrane, BCA assay kit, and the chemiluminescence kit were obtained from EMD Millipore (Billerica, MA, USA) and Thermo Fisher Scientific. The procedures were performed in strict accordance with the standard protocols. The Bradford method was used to ensure that the amount of protein per sample was 20 mg. The proteins were subjected to 10% SDS-PAGE on a Bio-Rad Mini PROTEAN 3 System (Bio-Rad Laboratories Inc., Hercules, CA, USA) and subsequently electrotransferred onto nitrocellulose membranes (400 mA for 2 h; EMD Millipore). Wet transfer was used for EGFR protein, while semi-dry transfer was used for VEGF, COX-2, and NF-κB. TBS containing 10% milk powder and 10% Tween-20 was used to block the nitrocellulose membranes at 37°C for 4 h. Then, the nitrocellulose membranes were incubated in primary antibodies overnight at 4°C. The column dilution ratio for all primary antibodies, including VEGF, COX-2, and NF-κB, but not β-actin (1:500; Santa Cruz Biotechnology Inc.), was 1:200. According to the appropriate primary antibody, we selected different rabbit or mouse IgG antibodies as the secondary antibodies. We selected an enhanced chemiluminescence (Thermo Fisher Scientific) detection system to detect light strips and then transferred the images to X-ray film (Del DOC2000; Bio-Rad Laboratories Inc.).
3
Protein Immunoblot Analysis of Irradiated Cells
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Immediately following irradiation, cells were lysed for 20 min at 4 °C in RIPA buffer (ThermoFisher Scientific) with protease and phosphatase inhibitors (#A32953 and #A32957, ThermoFisher Scientific). Lysates were centrifuged at 7000 RCF for 7 min at 4 °C. Proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (MilliporeSigma) as previously described [40 (link)]. nitrocellulose membranes were blocked in Tris-buffered saline with 5% BSA for 1.5 h. Primary antibodies were diluted in 5% BSA in Tris-buffered saline: β-actin (MilliporeSigma #A1982, 1:5000); DUSP1 (Cell Signaling #35217; 1:1000); total and phosphorylated p44/42 MAPK (Cell Signaling, Danvers, MA, USA, #4696 and #4370; 1:2000); total and Ser473 phosphorylated Akt (Cell Signaling #2920 and #4060; 1:2000); EGR1 (Cell Signaling #4154; 1:1000); full-length and cleaved caspase-3 (Cell Signaling #14220 and #9661; both 1:1000). Conjugated secondary antibodies (LI-COR, Lincoln, NE, USA; 1:10,000) were used for detection using the Odyssey system (LI-COR). β-actin was used as a loading control and for normalization of sample concentrations.
4
Protein Expression Analysis in Cardiac Cells
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H9c2 cells and mice hearts were freshly harvested and homogenized in RIPA lysis buffer (Beyotime, Beijing, China), sonicated for 20s, and normalized with the BCA Protein Assay Kit (Beyotime). The protein was separated by 10% SDS/PAGE and then electro-transferred onto nitrocellulose membrane (Millipore, MA, USA). After blocked with 5% non-fat milk for 2hrs, the nitrocellulose membrane was washed in Tris-Buffered Saline Tween-20 (TBS-T) for three 10mins and incubated with gentle agitation at 4°C overnight with the primary antibodies to PARP-1 (1:2000, sigma, CA, USA), ICAM-1 (1:500; Santa Cruz Biotechnology, CA, USA), iNOS (1:1000, cell signaling technology, MA, USA), cleaved caspase-3 (1:1000, cell signaling technology), cleaved caspase-9 (1:1000, cell signaling technology), IGF-1R (1:1000, cell signaling technology), p-IGF-1R (1:1000, cell signaling technology), Akt (1:1000, cell signaling technology), p-Akt (1:1000, cell signaling technology), nitrotyrosine (1:2000, cayman, MI, USA), β-actin (1:1000, Cell Signaling Technology) followed by washes with TBS-T and subsequent incubation with appropriate horseradish peroxidase-conjugated secondary antibody for 2hrs at room temperature. Labeled protein was visualized through enhanced chemiluminescence (Millipore).
5
Protein Extraction and Immunoblotting from Frozen Fish
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The frozen fish samples were pulverized with a mortar under liquid nitrogen and sonicated in a buffer containing 25% glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 20 mmol/L HEPES, 5 µmol/L DTT and 5 µmol/L PMSF at +4 °C. Protein extracts (30 µg protein per lane) were electrophoresed together with molecular weight markers on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore, Bedford, MA) [5] (link), [8] (link). Equal transfer was confirmed by reversible protein staining of the nitrocellulose membrane with Ponceau S (Sigma).
6
Recombinant Protein Expression Analysis
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The bacterial pellets were lysed by 50 μl 2x sample buffer (0.125M Tris-base pH 6.8, 4% SDS, 20% glycerol, 0.5% bromophenol blue, and 10% 2-Mercaptoethanol) and heated at 100°C for 10 minutes. Eventually, the prepared samples were collected, and the expression of the recombinant protein was analyzed by 4–12% SDS PAGE. The whole cell lysate proteins were transferred into nitrocellulose membrane (Sigma-Aldrich) for western blotting analysis by Trans-Blot SD Semi-Dry Transfer cell system (Biorad, USA). Subsequent to the western transfer of proteins, nitrocellulose membrane was blocked in TBS solution containing 4% skimmed milk to block nonspecific antibody binding overnight. Afterwards, the nitrocellulose membrane was washed with TBS and incubated with HRP conjugated mouse anti-polyhistdine monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) in 4% skimmed milk solution, 1:1000, for 1 hour at room temperature. Finally, the blotting reaction was performed, using diaminobenzidine (DAB)/H2O2 (Sigma-Aldrich, Poole, UK).
7
Optimized Reagent Procurement and Experimental Protocols
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Most of the material used in this study was purchased, as we reported earlier [38 (link),39 (link),40 (link),41 (link),42 (link),43 (link)]. Phosphate-buffered saline (PBS), nitrocellulose membranes, nicotine, BDNF, epinephrine, and propranolol hydrochloride were purchased from Sigma-Aldrich. The caspase 3 (cleaved) colorimetric In-Cell ELISA Kit (62218), Halt Protease and Phosphatase Inhibitor Cocktail, BCA protein assay kit, super signal west pico luminol (chemiluminescence) reagent, human IgG (hIgG) isotype control, α-tubulin monoclonal antibody (DM1A), goat anti-mouse IgG (H + L) superclonal secondary antibody, HRP conjugate (A28177), 3,3′,5,5′-tetramethylbenzidine (TMB), and lipofectamine 2000 transfection reagent were from ThermoFisher. Donkey anti-mouse IgG (HRP) (ab205724) was purchased from Abcam. MMP9 siRNA (sc-29400), MMP9 antibody (sc-393859), m-IgG Fc BP-HRP: sc-525409, and anti-E-cadherin antibody (DECMA-1, sc-59778) were from Santa Cruz Biotechnology. SignalSilence Control siRNA (unconjugated, 6568) was purchased from (Cell Signaling Technology, Danvers, MA, USA).
8
Quantifying Matrix Metalloproteinases by Western Blot
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According to the Laemmli method, all samples (10 μg of protein) were electrophoresed on SDS-polyacrylamide gel (10%) [29 (link)], blotted to nitrocellulose membranes (Sigma-Aldrich; Saint Louis, MO, USA) at 100 mA for 1 h. The membranes were blocked using 5% (w/v) nonfat powdered milk in the solution of TBS-T (20 mM Tris/HCl buffer, pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20) for 1 h. Then, samples were incubated overnight at 4 °C with antibodies against metalloproteinase-2 (Cat#MAB9021; R&D Systems, USA) or, respectively, directed against metalloproteinase-9 (Cat# MAB936; R&D Systems; USA) in TBS-T which contained 1% bovine serum albumin (w/v). In the next step, several washes in TBS-T buffer were performed. Bounded antibodies were detected using alkaline phosphatase (ALP) coupled with the appropriate antibody in the same solution at room temperature for 1 h with moderate shaking, and then BCIP/NBT reagent was used (Cat# B1911; Sigma; USA). Pre-stained molecular mass markers were used to determine the molecular mass of matrix metalloproteinases (BioRad, Hercules, CA, USA). Representative blots were demonstrated.
9
Western Blot Analysis of Apoptosis Regulators
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Cells were lysed using the mammalian protein extraction reagent RIPA (Beyotime, Shangahi, China), together with a protease inhibitor cocktail (Roche, Pleasanton, CA), and phenylmethylsulfonyl fluoride (Roche). Protein lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to 0.22 μm nitrocellulose membranes (Sigma–Aldrich), and incubated with specific antibodies against p53, mouse double minute 2 homolog (MDM2), Bcl-2, and Bcl-xl (Cell Signaling Technology, Danvers, MA). An anti-GAPDH antibody was used as a control. Signals were visualized using the ECL chromogenic substrate and quantified by densitometry using Quantity One software (Bio-Rad, Berkeley, CA).
10
Analysis of CD44 Protein Expression
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Cells were lysed using RIPA lysis buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing 10 mM NaF, and 0.5% protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA). Protein fractions were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes (Whatman, Sigma–Aldrich, St. Louis, MO, USA), blocked with 3% or 5% bovine serum albumin (BSA) in 1× TBST (Tris-buffered saline containing 0.1% Tween 20) for 1 h at room temperature, followed by overnight incubation with the rabbit polyclonal anti-CD44 antibody (Chemicon, Temecula, CA, USA), diluted in 1% BSA-TBST. After 3 washes with TBST the membranes were incubated for 1 h at room temperature with the infrared secondary antibody, anti-rabbit Dylight 680 (1:5000, Abcam, Cambridge, UK), diluted in 1% BSA-TBST and washed with TBST. The protein bands in the blots were visualized on a LI-COR Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).
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