12% SDS-PAGE and non-denaturing gradient polyacrylamide (4–20%, w/v) electrophoresis were carried out as described elsewhere. The IDH activity after electrophoresis was measured by incubating the gel slices in a solution that, stains based on the enzyme activity: 100 mM Tris-HCl buffer (pH 7.5), 2 mM MnCl2, 5.0 mM DL-isocitrate, 2.0 mM NAD+ or NADP+, 1.0 mM Nitroblue tetrazolium, and 0.5 mM phenazine methosulfate in 100 mM Tris-HCl buffer, pH 7.5. Isoelectric focusing experiments were performed in a horizontal slab gel. Pharmalyte 3–10 (GE Healthcare) was used to obtain a pH gradient.
Pharmalyte 3 10
Manufactured by GE Healthcare
Sourced in United States
Pharmalyte 3–10 is a laboratory equipment product used for isoelectric focusing (IEF) applications. It is a wide-range ampholyte that provides a continuous pH gradient from 3 to 10 for the separation and purification of proteins and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Urea, by Merck Group (2 mentions)
Iminodiacetic acid, by Merck Group (1 mentions)
Pngase f enzyme, by New England Biolabs (1 mentions)
L arginine, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Phosphoric acid, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Thiourea, by GE Healthcare (1 mentions)
Iodoacetamide, by GE Healthcare (1 mentions)
Ezq protein quantification assay, by Thermo Fisher Scientific (1 mentions)
Cydye, by GE Healthcare (1 mentions)
Readyprep 2 d cleanup kit, by Bio-Rad (1 mentions)
L lysine, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Acetonitrile acn, by Merck Group (1 mentions)
Ipgphor 2, by GE Healthcare (1 mentions)
Potassium persulfate, by Merck Group (1 mentions)
7 protocols using pharmalyte 3 10
1
Electrophoretic Analysis of IDH Activity
2
Amyloid-beta Species Isolation and Analysis
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7PA2 CM was concentrated ∼10-fold
using a Centriprep Ultracel YM-3 filter and then used for SEC as in
Figures1 B and 4 A. Fractions
8, 9, and 10 were pooled to a give a preparation rich in Aβ
immunoreactive species of masses between ∼14 and 4 kDa. This
material was then lyophilized, reconstituted in 125 μL of rehydration
buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, and 0.2% (v/v) Pharmalyte,
3–10) (GE Healthcare) and electrofocused on immobilized pH
gradient (IPG) strips (pH 4–7) (GE Healthcare). After focusing
was complete, IPG strips were incubated with 6 mL of SDS-equilibration
buffer (6 M urea, 50 mM Tris, 2% (w/v) SDS, 30% (v/v) glycerol, and
0.01% (w/v) phenol red) in a 15 mL tube for 15 min and then placed
on top of a 4–12% bis-tris gel. The IPG gel was secured in
position with agarose dissolved in running buffer so that the IPG
gel could be in contact the stacking gel. Molecular weight markers
were loaded in a well next to the IPG strip. Gels were electrophoresed
at 50 V for 10 min, and then the voltage was increased to 120 V for
2.5 h. The IPG strip was removed from the top of the gel and proteins
in the gel transferred onto nitrocellulose and Western blotted using
6E10 as described above and in Table1 .
using a Centriprep Ultracel YM-3 filter and then used for SEC as in
Figures
8, 9, and 10 were pooled to a give a preparation rich in Aβ
immunoreactive species of masses between ∼14 and 4 kDa. This
material was then lyophilized, reconstituted in 125 μL of rehydration
buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, and 0.2% (v/v) Pharmalyte,
3–10) (GE Healthcare) and electrofocused on immobilized pH
gradient (IPG) strips (pH 4–7) (GE Healthcare). After focusing
was complete, IPG strips were incubated with 6 mL of SDS-equilibration
buffer (6 M urea, 50 mM Tris, 2% (w/v) SDS, 30% (v/v) glycerol, and
0.01% (w/v) phenol red) in a 15 mL tube for 15 min and then placed
on top of a 4–12% bis-tris gel. The IPG gel was secured in
position with agarose dissolved in running buffer so that the IPG
gel could be in contact the stacking gel. Molecular weight markers
were loaded in a well next to the IPG strip. Gels were electrophoresed
at 50 V for 10 min, and then the voltage was increased to 120 V for
2.5 h. The IPG strip was removed from the top of the gel and proteins
in the gel transferred onto nitrocellulose and Western blotted using
6E10 as described above and in Table
3
Capillary isoelectric focusing (cIEF) protocol
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Sodium hydroxide, phosphoric acid, glacial acetic acid, urea, iminodiacetic acid, 2-mercaptoethanol and L-arginine were from Sigma Aldrich (St. Louis, MO, USA). The Pharmalyte 3–10 was from GE Healthcare (Chicago, IL, USA). The cIEF Gel; the pI markers of the pI Peptide Marker kit; the Fast Glycan kit; and the SDS-MW Analysis Assay kit including the SDS-MW Gel Buffer, Sample Buffer and 10 kDa protein standard along with the 50 µm ID NCHO and BFS capillaries were from Bio-Science Kft (Budapest, Hungary). The hIgG1 test sample was from Molecular Innovations (Novi, MI, USA). The commercial PNGase F enzyme was from New England Biolabs (Ipswich, MA, USA). The in-house produced 6His-PNGase F (production described in detail in [20 (link)] and this enzyme featured 3 months of shelf life) was from University of Pannonia (Veszprem, Hungary).
4
Quantitative Proteomic Sample Preparation
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Urea, ammonium bicarbonate and acetonitrile (ACN) were obtained from Merck (Darmstadt, Germany); thioUrea, CyDyes, iodoacetamide (IAA) and Pharmalyte 3–10 were obtained from GE Healthcare (Little Chalfont, UK); 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) was purchased from Roche Diagnostics (Basel, Switzerland). The EZQ protein quantification assay was from Life Technologies (Carlsbad, USA). Equilibration buffer and Electrode solutions for SDS-PAGE were obtained from Serva (Heidelberg, Germany). Dithiothreitol (DTT), Hydroxyethyldisulfide (HED), dimethylformamide (DMF), formic acid and L-lysine were purchased from Sigma-Aldrich (St. Louis, USA). The ReadyPrep 2D clean up kit was obtained from Bio-Rad (Hercules, USA). Sequencing grade modified trypsin was purchased from Promega (Finchburg, USA). All buffers were prepared using ultra-pure water from a Thermo Fisher Scientific system (Waltham, USA).
5
Optimized Glycan Analysis Protocol
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Sodium hydroxide (S2770-100ML), hydrocloric acid (1.09058.1000), sodium cyanoborohydride in 1 M tetrahydrofuran (296813-100ML), phosphoric acid (345245-100ML), urea (U0631-500G), iminodiacetic acid (220000-500G), iodoacetamide (I6125-5G) and L-arginine (A5006-100G) were purchased from Merck (Kenilworth, NJ, United States). Glacial acetic acid (02790-101-340) and acetonitrile (00630-517-350) were from Molar Chemicals (Halasztelek, Hungary). Glycerol (24388.295), tetrahydrofuran (28553.293) and 2-mercaptoethanol (M131-250ML) were from VWR (Radnor, PA, United States). The Pharmalyte 3-10 (17-0456-01) was from GE Healthcare (Chicago, IL, United States). The Advanced cIEF Starter Kit (A80976), the SDS-MW Analysis Kit (390953) and the Fast Glycan Kit (B94499PTO) were from Bio-Science Kft (Budapest, Hungary). The PNGase F enzyme and all exoglycosidase enzymes (Neuraminidase, Galactosidase and Hexosaminidase) were from the Bio-Nanosystems Laboratory, University of Pannonia (Veszprem, Hungary). The bamlanivimab (35 mg/ml; NDC 0002-7910-01) was a kind gift of the Borsod Academic County Hospital (Miskolc, Hungary).
6
Comprehensive 2D-PAGE Protein Analysis
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Eighteen 24 cm immobilized pH gradient (IPG) strips with a pH range of 3-10NL (Cat. No. 1632043, Bio-rad, Hercules, California, USA) were rehydrated in 500 μl of TUC1% (6 M urea, 2 M thiourea, 1.2% (v/v) 2,2 Dithiodiethanol (Cat. No. 380474, Sigma-Aldrich), 0.5% (v/v) Pharmalyte 3-10 (GE Healthcare) overnight at room temperature, then stored at −80°C until further use. 5μg of protein sample mixed with 5 μg IPS was loaded via anodal cup-loading. Isoelectric focusing (IEF) was carried out using an IPGPhor II (GE Healthcare) with following settings: 150 V for 1 h, 300 V for 1 h, 600 V for 1.5 h, increase to 8,000 V by gradient over 2 h, 24.000 Vh at 8,000 V, under exclusion of light. Current was limited to 50 μA per IPG strip. After IEF, IPG strips were stored at −80°C until further use. SDS-PAGE was carried out as described earlier (12 (link)).
7
Antioxidant Capacity Assay Protocol
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Bovine-serum albumin (BSA), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potassium persulfate, casein, cysteine, 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), N-α-carbobenzoxy-L-alanyl-p-nitrophenyl ester (ZANPE), and Tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma Chemical Company (St. Louis, MO, USA); Coomassie Brilliant Blue G-250 and R-250, acrylamide, bisacrylamide, tetramethylethylenediamine (TEMED), low-range molecular markers (Bio-Rad), from Bio-Rad (Hercules, CA, USA); trichloroacetic acid (TCA) from Carlo Erba Reagenti (Rodano, MI, Italy); Isoelectrofocusing (IEF) standards from Amersham, Pharmacia Biotech, UK; and ampholytes Pharmalyte 3-10 from GE Healthcare (Buckinghamshire, UK). All other chemicals were obtained from local commercial sources and were of the highest purity available.
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