Choline chloride (Salus Nutra Inc., Xian, China), DL-malic acid (Salus Nutra Inc., Xian, China), commercial chitin (Carbosynth, China), sucralfate (Combiphar, Bandung, Indonesia), ethanol, pure 99.5% (Merck Millipore-Sigma Aldrich, USA), ethyl ether (Merck Millipore-Sigma Aldrich, St. Louis, MI, USA), carboxymethyl cellulose (CMC) (BrataChem, Bandung, Indonesia), Bouin solution, distilled water (BrataChem, Bandung, Indonesia), mouse monoclonal IgG1 NF-kappaB p65 antibody (Santacruz Biotechnology Inc., K2320, Shanghai, China), β-actin antibody (Thermo Scientific MA5-15739, Singapore), anti-mouse antibody (Li-Cor C90408-07, PT. Elo Karsa Utama, Jakarta, Indonesia), bovine serum albumin (BSA Protein, Thermo Scientific, Singapore), sodium dodecyl sulfate (SDS) gel 15% (Merck Millipore-Sigma Aldrich, St. Louis, MI, USA), polyvinylidene difluoride (PVDF) membrane (Merck Millipore-Sigma Aldrich, St. Louis, MI, USA), phosphate buffer saline-tween 20 (PBST) 0.1%, protein ladder (Thermo Scientific, Singapore), running buffer, transfer buffer, lysis buffer.
Protein ladder
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania, China, United Kingdom
A protein ladder, also known as a molecular weight marker, is a mixture of pre-stained protein standards with known molecular weights. It is used as a reference to determine the approximate molecular weight of proteins separated by gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (5 mentions)
Trypsin, by Merck Group (3 mentions)
Ecl substrate, by Bio-Rad (3 mentions)
Glycerol, by Merck Group (3 mentions)
Acrylamide, by Merck Group (3 mentions)
Glycine, by Merck Group (3 mentions)
Bromophenol blue, by Merck Group (3 mentions)
Tetramethylethylenediamine, by Merck Group (3 mentions)
Ammonium persulphate, by Merck Group (3 mentions)
Sodium dodecyl sulfate (sds), by Bio-Rad (3 mentions)
Bis acrylamide, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Pha l, by Merck Group (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (2 mentions)
Hematoxylin, by Solarbio (2 mentions)
Page gel silver staining kit, by Solarbio (2 mentions)
Dab substrate kit, by ZSGB-BIO (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
63 protocols using protein ladder
1
Cellular Signaling Pathway Evaluation
2
SDS-PAGE Analysis of Leaf Proteins
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Leaves of all cultivars were collected from 8-week old plants grown under control (T1), one level of salt stress (6 g L−1 NaCl) (T3), two levels of ZnO-NPs (15 (T4) and 30 (T5) mg L−1) and the combinations between NaCl and ZnO-NPs (6 g L−1 NaCl + 15 mg L−1 ZnO-NPs (T8); 6 g L−1 NaCl + 30 mg L−1 ZnO-NPs (T9)) and stored at −80 °C until protein analysis. Briefly, 0.5 g of frozen leaf tissue was used to extract soluble protein according to Bradford (1976) . SDS–PAGE of leaf protein extracts were carried out in a vertical slab gel using 12% acrylamide according to Laemmli (1970) (link) and a volume of 15–20 μL was applied to each well. In a separate lane of the gel, a protein ladder ranging from 10 to 250 kDa (Thermo Fisher Scientific, Waltham, MA, USA) was loaded in order to allow the estimation of the molecular masses of the separated proteins. Electrophoresis was run in a protein II electrophoresis system (Bio-Rad, California, USA) for about one hour in running buffer at 150 V/100 mA. The gels obtained were photographed with a gel documentation system (Syngene, Cambridge, UK). The molecular weights of the dissociated or unknown protein bands were determined using the standard curve obtained from the Rf-values and molecular weights of the protein ladder (10–250 kDa) and calculated using the gel analyzer version 3 software program.
3
Purification and Western Blot Analysis of SARS-CoV-2 Spike Protein
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An aliquot comprising 7 mL of pseudotyped virus solution was added to a 2 mL 25% sucrose buffer and centrifuged at 100,000 × g for 3 h. The purified virus particles were then re-suspended in 100 μL of PBS buffer, and 20 μL of 6× SDS sample buffer was added to 100 μL of the re-suspended pseudotyped virus, mixed well, and heated in a 100 °C metal bath for 5 min. Then, a 15 μL sample was used for SDS-PAGE and western blot analysis. The protein ladder (Thermo Fisher Scientific, Cat: 26619) was loaded as a molecular weight marker.
A 500-fold dilution of SARS-CoV-2 (2019-nCoV) spike antibody was used as the primary antibody. The polyclonal antibody was harvested 7 days after thrice immunization of SPF balb/c mice with purified S2 protein. A 1:10,000 dilution of HRP-conjugated goat anti-mouse IgG (CW Biotech) was used as the secondary antibody. The VSV M protein was blocked as an internal reference using VSV-M antibody (23H12, Cat: EB0011, kerafast). Immobilon Western chemiluminescent HRP substrate (Millipore) was used to develop the immune-reactive bands.
A 500-fold dilution of SARS-CoV-2 (2019-nCoV) spike antibody was used as the primary antibody. The polyclonal antibody was harvested 7 days after thrice immunization of SPF balb/c mice with purified S2 protein. A 1:10,000 dilution of HRP-conjugated goat anti-mouse IgG (CW Biotech) was used as the secondary antibody. The VSV M protein was blocked as an internal reference using VSV-M antibody (23H12, Cat: EB0011, kerafast). Immobilon Western chemiluminescent HRP substrate (Millipore) was used to develop the immune-reactive bands.
4
Extraction and Characterization of FX from Brown Algae
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FX was extracted and refined from brown algae, as described previously [48 (link)], and prepared and identified in our laboratory (purity ≥ 50%) [49 (link),50 (link)]. Analytically pure sodium borohydride, lithium aluminum hydride, sodium hydroxide, and sodium bicarbonate were obtained from National Pharmaceutical Holding Chemical Reagent Co., Ltd. (Shanghai, China). All enzymes, such as porcine pancreatic lipase, Pseudomonas cholesterol esterase, and Candida rugosa lipase, were obtained from Sigma-Aldrich (St Louis, MO, USA). Lipase (Brand: Kramer) was obtained from Ziyi Reagent Factory (Shanghai, China). The protein ladder was acquired from Thermo Fisher (Waltham, MA, USA). Escherichia coli strain BL21 (DE3) was preserved in our laboratory. HPLC-grade methanol was obtained from Merck KGaA (Darmstadt, Germany). Ultrapure water was produced using a Millipore Milli-Q system (Millipore Corp., Billerica, MA, USA). The chemical reagents used in this research were of analytical grade and purchased from Sigma-Aldrich (Saint Louis, MO, USA).
5
Western Blot Analysis of Protein Modifications
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Cells and tissues were collected and dissolved in RIPA lysis buffer (Beyotime Biotechnology) and the proteins were extracted according to the manufacturer's protocol. Proteins were separated via 12% SDS-PAGE and transferred to nitrocellulose filters (EMD Millipore). The membranes were cut into small pieces according to the protein ladder (Thermo Scientific, Inc.) and the parts with the target proteins were reserved. After that, the membranes were blocked with 5% non-fat milk in TBST buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl and 0.05% Tween-20) for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: H2A.X (dilution 1:500), NPM1 (dilution 1:500), pan-acetylation (dilution 1:1,000), pan-succinylation (dilution 1:1,000), GAPDH (dilution 1:1,000). The membranes were then washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody (dilution 1:2,500) for 30 min at room temperature. After extensive washing, the proteins were detected using an Electrochemiluminescence-Plus kit (CoWin Biotechnology) and imaged using a Gel-imaging system (Bio-Rad) with Image Lab Software (version 5.2; Bio-Rad Laboratories).
6
Investigating Metabolic Modulators and Antioxidants
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2-Ketoglutaric acid (α-KG, CAS#328-50-7), EGCG (A606599), and N-acetyl-L-cysteine (NAC, CAS#616-91-1) were purchased from Sangon Biotech. BPTES (SML0601) and 2′,7′-dichlorofluorescein-diacetate (DCFH-DA, HY-D0940) were purchased from Sigma-Aldrich. DMEM was purchased from Hyclone. VH-298 (HY-100947) was purchased from MCE. The following antibodies were used for western blot: Sirt4 (sc-135797, Santa), HO-1 (sc-136960, Santa), HIF-1 alpha (PA1-184, Invitrogen), caspase-9 (CASP9) (sc-56076, Santa), Bax (50599-2-ig, Proteintech), Bcl-2 (12789-1-AP, Proteintech), and protein ladder (Thermo Fisher #26616).
7
Immunohistochemistry and Cell Assays
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PHA-L (L2769) was purchased from Sigma (Saint Louis, MO, USA). PAGE Gel Silver Staining Kit (G7210) was purchased from Solarbio (Beijing, China). Cell Counting Kit-8 (CK04) was from Dojindo (Kumamoto, Japan), and CytoTox 96 Non-radioactive Cytotoxicity Assay (G1782) was from Promega (Madison, WI, USA). In Situ Cell Death Detection Kit-TMR red was from Roche Applied Science (12156792910, Mannheim, Germany). Protein ladder was purchased from Thermo (26616), (Vilnius, Lithuania). DAB substrate kit was from ZSBIO (ZLI-9017, Beijing, China), and hematoxylin was from Solarbio (G4070). All the antibodies against CD3E (512415), CD8A (510793), Granzyme B (252579), and Perforin (500093) were purchased from ZEN BIO (Chengdu, China). The A549 (human non-small cell lung cancer cells), Jurkat (human acute T cell leukemia cells), B16-F10 (mouse melanoma cells) and YAC-1 (mouse lymphoma cells) cell lines were from American type culture collection (ATCC).
8
Immunophenotyping and Western Blot Analysis
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Immunophenotyping was performed by flow cytometry, and related antibodies were purchased from Dako Denmark A/S (Glostrup, Denmark); Lymphoprep™, RNX-Plus and cDNA synthesis kits were from Axis-Shield (Oslo, Norway), Cinnagen (Tehran, Iran) and Fermentas (Waltham, MA, USA). Real-time PCR kit was purchased from TaKaRa (Otsu, Japan); RIPA lysis buffer was from Sigma-Aldrich (St Louis, MO, USA) and protease inhibitor cocktail was purchased from Melford (Melford, UK). Protein ladder and PVDF blotting membrane were from Thermo Fisher Scientific (Waltham, MA, USA) and GE Healthcare (Buckinghamshire, UK), respectively. Goat anti-ABCA2 polyclonal antibody and goat anti-ABCA3 polyclonal antibody were purchased from Santa Cruz Biotechnology Inc., (Dallas, TX, USA); rabbit anti-goat IgG-HRP was from Sigma-Aldrich and mouse anti-GAPDH monoclonal antibody was purchased from EMD Millipore (Billerica, MA, USA). Goat anti-mouse IgG-HRP was purchased from Dako Denmark A/S and ECL solution was from Bio-Rad Laboratories Inc., (Hercules, CA, USA).
9
Gelatin Zymography for MMP-2/9 Assessment
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A gelatin zymography kit (Cosmo Bio Co., LTD, cat. n. PMC-AK47-COS, Tokyo, Japan) was used to determine the gelatin degrading activity of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in sera of 29 DMD patients. Before performing the gelatin zymography, total serum protein concentration was determined with the BCA protein assay kit (Thermo Fisher Scientific, cat. n. 23225, Etten-Leur, the Netherlands). Serum samples were diluted 50-fold in distilled water and the concentrations were measured in triplicates according the manufacturer’s instructions. Final protein concentrations were adjusted to 2 µg/µl and 20 µg (10 µl) of total protein was mixed with 10 µl of sample preparation buffer, incubated for 15 minutes at room temperature and loaded onto the provided gel. Ten µl of the MMP marker and 5 µl of a protein ladder (Thermo Fisher Scientific, cat. n. 26616, Etten-Leur, the Netherlands) were loaded as controls. The electrophoresis was run in a XCell SureLock™ Mini-Cell Electrophoresis System (Life Technologies, Carlsbad, CA, USA) at 150 mA for 2 hours. Latent MMP forms were activated by incubation with a reaction buffer, the gel was stained with the staining solution provided by the kit manufacturer and scanned using an Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, USA). Band intensities were quantified using ImageJ.
10
Western Blotting for GluA1 Subunits
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A total of 100 μl lysate solution, sample buffer (0.3 M Tris-HCl, 10% SDS, 50% glycerol, 0.25% bromophenol blue, 0.5 M dithiothreitol), and dH2O were prepared and boiled for 2 min at 100 °C. Samples and a protein ladder (Thermo Scientific) were loaded into a 7.5% SDS-PAGE gel. Sample separation by SDS-PAGE was followed by transference to a nitrocellulose membrane (Millipore). Membranes were cut horizontally at the 72 kDa level, and the upper portion was probed with a rabbit antibody for GluA1 subunits (1:7000, Cell Signalling)40 (link), and the lower portion was probed for β-actin (1:5000, Cell Signalling). Membranes were incubated in primary antibody overnight at 4 °C on a shaker. Next day membranes were washed 3 × 5 min with 1X TBST. HRP-bounded 2nd antibodies were applied (1:10,000, anti-rabbit; Pierce) for 1 hr. Membranes were then washed 3 × 10 min in TBST and enhanced chemiluminescence Western blotting substrate (Pierce) was applied. Blots were then developed on x-ray film (AGFA). Films were scanned using an image scanner (CanoScan LiDE 200), and the optical density (OD) of each band was measured using ImageJ software. Each sample was normalized to the corresponding β-actin band that was run on the same gel.
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