BDNF, leupeptin, aprotinin, phenyl-methylsulfonyl fluoride, pepstatin A, soybean trypsin inhibitor, NaF, sodium vanadate, glycerophosphate, 2-mercaptoethanol, NMDA, glycine, Tween 20, NP-40, and Histopaque-1077 were from Sigma. Anti-TrkB (SC-8316), -pY-Trk (SC-8058), -phospholipase C-γ1 (SC-7290), -N-Shc (SC-365598), -NR1 (SC-9058), -TNFα (SC-52746), -IL-6 (SC-57315), -IL-1β (SC-57315), -Arc (SC-365736), - BDNF/proBDNF (SC-2098), –NT3 (SC-547), -NT4 (SC-545), -β-actin (SC-47778) were from Santa Cruz Biotechnology. FNDC-5/irisin (36–335) was from ProSci (Poway, CA, USA). Seize-X immunoprecipitation kit, antigen elution buffer, Bind NeutrAvidin, high binding capacity coated 96-well plates, and West Pico chemiluminescent reagents were from Pierce-Endogen (Rockford, IL, USA). Bradford reagent, SDS-PAGE reagents, and pre-stained molecular weight markers were from Bio-Rad. Protease inhibitors (EDTA-free) and protein phosphatase inhibitor tablets were from Roche (Basel, Switzerland). BDNF was reconstituted according to the manufacturer’s instruction. To avoid freezing damage, 10% glycerol was added for a final concentration of 10 ng/μl BDNF and stored in 80°C until use. All other test agents were made fresh according to the manufacturer’s recommendation. The DMSO concentration in the incubation medium was 1%.
Prestained molecular weight markers
Manufactured by Bio-Rad
Sourced in United States
Prestained molecular weight markers are laboratory reagents used to estimate the molecular weights of proteins in electrophoresis experiments. They contain a mixture of pre-stained proteins with known molecular weights, which can be used as size references when analyzing protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Sds polyacrylamide gel, by Bio-Rad (2 mentions)
Bradford protein assay, by Merck Group (2 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (2 mentions)
Gradient polyacrylamide gel, by GenScript (2 mentions)
Ibright cl1000, by Thermo Fisher Scientific (2 mentions)
Pepstatin a, by Merck Group (1 mentions)
Soybean trypsin inhibitor, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Tween 20, by Merck Group (1 mentions)
Brain derived neurotrophic factor (bdnf), by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Np 40, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Il 1β, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Bdnf probdnf, by Santa Cruz Biotechnology (1 mentions)
22 protocols using prestained molecular weight markers
1
BDNF Signaling Protein Detection
2
Western Blot Analysis of Protein Signaling
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Samples were denatured in buffer containing 60 mM Tris/pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate (SDS), and 14.4 mM 2-mercaptoethanol with 0.1% bromophenol blue, and boiled for 5 minutes. SDS polyacrylamide gels (Bio-Rad, Hercules, CA, USA) were loaded with 25 µg of total protein per lane. Prestained molecular weight markers (Bio-Rad) were used as standards. Electrophoresed samples were transferred to polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ, USA). After transfer, membranes were blocked with 3% bovine serum albumin in Tris-phosphate-buffered saline (TPBS; 200 mM Tris/pH 7.0, 1.37 M NaCl, 1% Tween-20) for 1 hour at room temperature. Membranes were then incubated with anti-RET, anti-phospho-RET, anti-EGFR, anti-phospho-EGFR, anti-AKT, anti-phospho-AKT, anti-ERK, anti-phospho-ERK, or anti-beta actin (1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4℃, washed as before, and incubated with goat anti-rabbit peroxidase-conjugated secondary antibody (1:4000; Cell Signaling Technology) for 2 hours at room temperature. Membranes were then washed, and expressed proteins were detected with Pierce ECL plus western blotting substrate (Pierce, Rockford, IL, USA). Experiments were conducted at least three times independently.
3
Quantitative Analysis of Macrophage Proteins
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CM collected from human macrophage cell culture were analyzed for TLR-2, TLR-4, and MMP-9 by Western blot analysis. All Western blot procedures were performed as described by us previously [22 (link)]. Prestained molecular weight markers were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). CM samples were electrophoresed on SDS-PAGE (8% separating and 4% stacking gels). Proteins were transferred to nitrocellulose membranes, and incubated with primary antibodies (TLR-2: Abcam, Cambridge, MA; TLR-4: Life Technologies, Carlsbad, CA; MMP-9: Cell Signaling Technology, Danvers, MA). Excess primary antibodies were removed before incubation with the secondary antibodies conjugated to horseradish peroxidase (Goat anti-rabbit antibodies, Cell Signaling Co., Danvers, MA). Detection of the protein bands was carried out by scanning the membranes with Invitrogen™ iBright™ FL1000 Western Blot Imaging Systems (Thermo Fisher Scientific, Inc., USA) and quantified by measuring band intensity using Image J analysis software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
4
Western Blot Analysis of Proteins
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Protein samples were resolved on SDS-polyacrylamide gels and transferred to Hybond-P PVDF membranes (Amersham). Membranes were blocked for 1 h in TBS, 0.1% Tween 20 and 5% non-fat dry milk, followed by an overnight incubation with primary antibodies (see Materials and Methods) diluted in the same buffer. After washing with 0.1% Tween in Tris-buffered saline, the membrane was incubated with peroxidase-conjugated secondary antibody for 1 h, and then washed and developed using the ECL chemiluminescent detection system (Roche). Densitometric analyses were performed using ImageJ software program (National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/ij/ ) and normalized against the signal obtained by reprobing the membranes with mouse anti-β-actin or anti-tubulin. The apparent molecular weight of proteins was determined by calibrating the blots with prestained molecular weight markers (Bio-Rad Laboratories, Richmond, CA, USA).
5
Quantitative Western Blot Analysis
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Protein content was determined using Bradford protein assay (27813, Sigma-Aldrich, Merk KGaA). Uniform amounts of protein extracts were loaded by standard SDS/PAGE. Samples were then electroblotted on Pierce Nitrocellulose Membrane, 0.45 µm (Thermo Fisher Scientific). Then, membranes were incubated in a blocking solution of 3% low-fat milk, diluted in PBS 1X-Tween 0.05% solution with the indicated primary antibody for 16 h at 4°C. Secondary antibody were used to reveal immunocomplexes using LiteUP WB Chemiluminescent Substrate (EMP002005, Euroclone, Hercules, California, USA). Chemiluminescent imaging was obtained using ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, California, USA). The apparent molecular weight of proteins was determined by calibrating the blots with pre-stained molecular weight markers (Bio-Rad Laboratories).
6
Western Blot Analysis of A1 Adenosine Receptor
7
UV Cross-linking of DNA-Protein Complexes
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Ultraviolet (UV) cross-linking of DNA/protein complexes was carried out essentially as described (13 (link)). Bromodeoxyuridine (BrdU)-substituted 32P-labeled probes were prepared by annealing the oligonucleotide primer UV-common (5′-GATCCACTGAGCCT-3′) with the E2F-c-myb-UV oligonucleotide (CTAGACAGATTTGGC GGGAGGGGGAGGCTCAGTG-3′). Labeling (200 ng) was achieved by a Klenow fill-in reaction performed for 1–2 h at 4°C with [α-32P]dGTP and [α-32P]dCTP and unlabeled dATP and bromo-dUTP (Sigma-Aldrich) in the labeling reaction. Unincorporated nucleotides were removed by chromatography over Sephadex G25. Electrophoretic mobility shift analyses (EMSAs) were performed with the BrdU-substituted probes as described above except that the binding reaction was scaled-up 2-fold. Immediately after electrophoresis, the wet gel was covered with plastic wrap and exposed to UV light (500 mJ) with a Stratalinker 1800 (Agilent Technologies, Santa Clara, CA, USA). The positions of various complexes were determined by 1–2 h autoradiography at 4°C. Gel slices containing various complexes were excised and soaked in 50–100 μl of 2× sodium dodecyl sulfate (SDS) sample buffer at 65°C for 30 min. The samples were boiled for 5 min and electrophoresed through an SDS-8% polyacrylamide gel along with pre-stained molecular weight markers (BioRad Laboratories, Hercules, CA, USA).
8
Recombinant Streptolysin O Protein Purification and Characterization
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Recombinant streptolysin O (SLO) was obtained from Dr. Bhakdi (University of Mainz, Mainz, Germany). The rabbit polyclonal antibody directed towards human α-synuclein (purified on protein A) was from Axxora, LLC (Farmingdale, NY). The mouse monoclonal anti-synaptobrevin-2 antibody (clone 69.1, purified IgG) and the rabbit polyclonal anti-complexin I/II (purified IgG) were from Synaptic Systems (Göttingen, Germany). HisTrap columns, FF, Cytiva (formerly GE healthcare Life Sciences) were purchased from ALLSCIENCE, LLC (Doral, FL). FITC-coupled Pisum sativum agglutinin (FITC-PSA) was from Vector Labs (BIOARS S.A, Buenos Aires, Argentina). PSA coupled to 20 nm colloidal gold was from glycoMatrix (Dublin, OH). Horseradish peroxidase- and Cy™3-conjugated goat anti-rabbit, as well as Cy™3-conjugated goat anti-mouse IgGs (H + L) were from Jackson ImmunoResearch (West Grove, PA). O-nitrophenyl EGTA-acetoxymethyl ester (NP-EGTA-AM) was purchased from Life Technologies (Buenos Aires, Argentina). Ni-NTA-agarose was from GE Healthcare. Prestained molecular weight markers were from Bio-Rad (Tecnolab, Buenos Aires, Argentina). All other chemicals were from Sigma-Aldrich™ Argentina S.A., Genbiotech, or Tecnolab (Buenos Aires, Argentina).
9
Recombinant LTB and CTB Protein Production
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Murashige and Skoog (MS) medium, spectinomycin, and sucrose were purchased from MB cell (CA, U.S.A). Tetracycline, cefotaxime and phosphinothricin were purchased from Duchefa (Haarlem, Netherlands). The pMJ103 vector was constructed by GreenGene BioTech (Yongin, Korea) and the plasmids (MYO51, MYO53) containing LTB and/or CTB were kindly donated from Dr. M.S. Yang (Chonbuk National University). T4 DNA ligase and restriction endonucleases were obtained from Roche (Basel, Switzerland). Gateway BP and LR recombinase systems were purchased from Invitrogen (Breda, Netherlands). A mini trans-blot cell and Mini-Protean III cell were purchased from Bio-Rad (Hercules, CA, USA). Reagents for SDS-polyacrylamide electrophoresis, such as acrylamide, bis-acrylamide, ammonium persulfate, TEMED, prestained molecular weight markers and Coomassie Brilliant Blue R-250 solution were obtained from Bio-Rad (Hercules, CA, USA). Anti-LTB polyclonal antibody was obtained from Abcam (Cambridge, MA, USA). GM1-ganglioside and anti-rabbit IgG-conjugated alkaline phosphatase were purchased from Santa-Cruz Biotechnology (CA, USA). Thiamine-HCl, myo-inositol, anti-CTB polyclonal antibody, and other reagents such as salts and buffer components were of analytical grade and purchased from Sigma (St. Louis, MO, USA).
10
Western Blot and Zymography Analysis
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Western blots and zymography were performed as described [9 (link),11 (link)]. Briefly, proteins (40 μg/sample) in sodium dodecyl sulfate (SDS)-loading buffer were electrophoresed through 10–18% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes. Membranes were incubated with specific antibodies against PARP-1 (#9542, and #5625, Cell Signaling Technology). After development, membranes were stripped and reblotted with anti-actin antibody (Santa Cruz Biotechnology).
gelatinolytic activity was detected in liver extracts (80μg) by 10% SDS-PAGE contained 1mg/ml of gelatin (Invitrogen), under non-reducing conditions. After incubation in renaturation buffer (Bio-Rad) and development buffer (50 mmol/L Tris-HCl, 5 mmol/L CaCl2, and 0.02% NaN3, pH 7.5), gels were stained with Coomassie brilliant blue R-250 (Bio-Rad), and destained with methanol/acetic acid/water (20:10:70). Prestained molecular weight markers (Bio-Rad), MMP-2, and MMP-9 (BIOMOL International) served as standards. Relative quantities of protein were determined using a densitometer (NIH Image J software)
gelatinolytic activity was detected in liver extracts (80μg) by 10% SDS-PAGE contained 1mg/ml of gelatin (Invitrogen), under non-reducing conditions. After incubation in renaturation buffer (Bio-Rad) and development buffer (50 mmol/L Tris-HCl, 5 mmol/L CaCl2, and 0.02% NaN3, pH 7.5), gels were stained with Coomassie brilliant blue R-250 (Bio-Rad), and destained with methanol/acetic acid/water (20:10:70). Prestained molecular weight markers (Bio-Rad), MMP-2, and MMP-9 (BIOMOL International) served as standards. Relative quantities of protein were determined using a densitometer (NIH Image J software)
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