The identification of lysines in heparin binding sites (HBS) was according to Ori et al. [34 (link)] with minor modifications. A heparin minicolumn was made by placing a plastic air filter at the end of a small pipette tip into which 30 µl of AF-heparin beads (Tosoh Biosciences, Stuttgart, Germany; binding capacity 4 mg antithrombin III ml−1 resin) was packed. A 5 ml syringe was used to pack the minicolumn and dispense buffer. The heparin column was equilibrated four times with 50 µl of PB 150 buffer (17.9 mM Na2HPO4, 2.1 mM NaH2PO4, 150 mM NaCl, pH 7.8). A minimum of 40 µg FGF protein was loaded onto the heparin column, and the loading was repeated three times with the same sample. After binding, the column was washed with PB 150 buffer four times. To acetylate exposed lysines, the minicolumn was then quickly rinsed with 20 µl of PB 150 containing 50 mM sulfo-NHS–acetate (Life Technologies, Paisley, UK) and then incubated for 5 min with 20 µl of fresh PB 150 containing 50 mM sulfo-NHS–acetate at room temperature. After acetylation, the minicolumn was washed with 50 µl of PB 150 buffer, and acetylated protein was eluted from heparin with 2 × 20 µl elution buffer (45 mM Na2HPO4, 5 mM NaH2PO4, 2 M NaCl, pH 7.8).
Sulfo nhs acetate
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Sulfo-NHS-acetate is a water-soluble, N-Hydroxysuccinimide (NHS) ester that can be used for chemical modification of proteins and other molecules containing primary amine groups. It provides a convenient method for introducing acetyl groups onto amine-containing compounds.
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Lab products found in correlation
P hydroxyphenylglyoxal, by Thermo Fisher Scientific (2 mentions)
Sp 495, by R&D Systems (1 mentions)
Stressmarq, by Thermo Fisher Scientific (1 mentions)
Centrifugal filter, by Merck Group (1 mentions)
Q125 sonicator, by Qsonica (1 mentions)
Thermolysin from geobacillus stearothermophilus, by Merck Group (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Molecular imager gel doc xr system, by Bio-Rad (1 mentions)
Pageruler plus prestained ladder, by Thermo Fisher Scientific (1 mentions)
Biotin teg phosphoramidite, by Glen Research (1 mentions)
Akta basic system, by GE Healthcare (1 mentions)
Inova 500, by Bruker (1 mentions)
Human α thrombin, by Prolytix (1 mentions)
Thrombin cleancleave kit, by Merck Group (1 mentions)
Blocker bsa in tbs, by Thermo Fisher Scientific (1 mentions)
10 protocols using sulfo nhs acetate
1
Identification of Lysines in Heparin Binding Sites
2
Peptide Modification and Purification
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Peptides were purchased from China Peptide (China) and Synpeptide (China). Both batches were purified to >95% purity by HPLC and validated by mass spectrometry (See Supplementary Data S2 for Synpeptide quality control data). No variation in peptide activity was observed between batches. Each batch was reconstituted in water and the concentration adjusted to 100 µM based on spectroscopy using 41480 (N1) and 35980 (N2) extinction co-efficients. To block the basic charged residues of N1, sulfo-NHS acetate (Thermo Scientific) was reacted with primary amines by incubation of equal masses of peptide and sulfo-NHS acetate in PBS for 1 hour at room temp. Reacted peptides were washed 3 × in 3 kDa cut-off spin columns in PBS, then resuspended in PBS to a final concentration of 100 µM. This reaction produced extensive modification of the peptide (Supplementary Figure S3 ).
3
Chemical Modification of Proteins
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Chemical acetylation was performed with sulfo-NHS-acetate (Thermo Scientific, Rockford, IL) as described [10 (link)]. Succinylation was done with sulfo-NHS-succinate which was prepared as follows. 9.7 mg of sodium succinate, 7.8 mg of NHS, and 9.7 mg of EDC were dissolved in 300 μl of dimethylformamide in a glass vial and incubated at room temperature for 16 hr. From this reaction, 50μl was removed to a new vial, dried under nitrogen, and washed with ether. After re-drying, the material was dissolved in 50 μl of water and incubated immediately with VLCAD protein for 2 hr at room temperature. All acylated proteins were dialyzed overnight prior to experimentation.
4
Fluorescent Labeling of α-Synuclein Proteins
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Commercial recombinant proteins, including human Tau (R&D Systems, SP-495), α-Syn (Proteos, cat# RP-003), oligomeric α-Syn (StressMarq, cat# SPR-484), α-Syn preformed fibrils (PFFs) (StressMarq, cat# SPR-322-C), α-Syn N (1–60) fragment (rPeptide, cat# S-1011–1), and α-Syn ΔN (61–140) fragment (rPeptide, cat# S-1013–1) were labeled with Alexa Fluor® 488 ester (Life Technologies, cat# A10235) according to the manufacturer’s instructions. After labeling, 100 mM glycine was added to quench the reaction and the proteins were subjected to Amicon Ultra-0.5 mL Centrifugal Filters (Millipore, cat# UFC500396 -3 KDa, cat# UFC501096 -10 KDa, and cat# UFC510096 -100 KDa) to remove any unreacted label. The fluorescently labeled α-Syn PFFs were sonicated using a Qsonica Q125 Sonicator at 30% amplitude for 30 cycles (1 s ON, 1 s OFF) before incubating with cells. Lysine capped proteins were prepared with Sulfo-NHS-Acetate (Thermo, cat# 26777) according to the manufacturer’s instructions.
5
Peptide Labeling and Quantification
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NAC N-terminal peptides were ordered from Genscript. Peptides were dissolved to 1 mM in 0.1 M sodium carbonate buffer, pH 8.5. Peptide solutions were added to a 25-fold molar excess of Sulfo-NHS Acetate (Thermo Scientific, Waltham, MA, USA) to amine groups in the sample and incubated for 1 hour at room temperature. Reaction was quenched using a 1 M Tris-HCl, pH 7.5 solution. To quantify the efficiency of this labeling, a lysine standard curve was established; samples were assayed for free primary amines by adding 0.01% (w/v) TNBS in 0.1 M sodium bicarbonate, pH 8.5 solution (G Biosciences) to each sample and standard, and incubated at 37°C for 2 hours. 10% SDS and 1N HCl was then added to stop the reaction. Absorbance was measured at 335 nm.
6
Chemical Modification of Beta-Amyloid Peptide
7
Acetylation and Cy5-Labeling of Thermolysin
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Thermolysin from Geobacillus stearothermophilus (Sigma-Aldrich) was acetylated using a 10:1 w/w surplus of sulfo-NHS-acetate (Thermo Fisher Scientific) for half an hour at room temperature in 100 mm triethylammonium bicarbonate (TEAB), pH 8.3. The reaction was quenched with 50 mm Tris-HCl, pH 8, and the acetylated thermolysin was desalted into HBS with 5 mm CaCl2 using a PD-10 column (GE Healthcare).
Cy5-labeled thermolysin was prepared by labeling thermolysin with a 1:1 molar ratio of Cy5 mono NHS ester (GE Healthcare) in 100 mm TEAB, pH 8.3, for half an hour at room temperature. The reaction was quenched with Tris-HCl and the Cy5-labeled thermolysin was desalted as for acetylated thermolysin. Absorbance at 280 nm and 550 nm was used to calculate the labeling ratio, which was an ∼0.6:1 molar ratio of Cy5 to thermolysin. A portion of the Cy5-labeled thermolysin was then acetylated as described for unlabeled thermolysin.
Cy5-labeled thermolysin was prepared by labeling thermolysin with a 1:1 molar ratio of Cy5 mono NHS ester (GE Healthcare) in 100 m
8
SDS Stability Assay for Protein Analysis
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The SDS stability assay is based on previously reported assays [35] (link), [36] (link). To 55 µM protein in pH 7 buffer either a 10-fold molar excess of biotin-5-fluorescein or the same volume of pH 7 buffer was added. Due to the high isoelectric point of avidin, A/A2-1, A/A2-B and A/A2-B I109K (pI>9), the proteins (including AVR2, pI = 4) were acetylated by adding a 40-fold molar excess of Sulfo-NHS-acetate (Thermo Scientific) [35] (link). The protein sample was divided into equal aliquots and incubated for 20 min at 20, 40, 50, 60, 70, 80, 90, or 100°C. After incubation, the same volume of 2× sample buffer (60 µM Tris-HCl, pH 6.8, 25% glycerol, 0.01% Bromphenol blue) was added, before loading 5 µg of the protein in a 15% acrylamide-bisacrylamide gel. To detect the bound biotin-5-fluorescein, the gels were analyzed under UV in a Molecular Imager Gel Doc XR+ System (BioRad Laboratories, Hercules, CA, USA), before Coomassie Brilliant Blue staining. As a control, proteins were incubated in denaturizing SDS sample buffer (60 µM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% Bromphenol blue, 0.5% β-mercaptoethanol) for 20 min at 100°C. PageRuler Plus Prestained Ladder (Thermo Scientific) was used as molecular weight marker.
9
Thrombin-Binding Aptamer Synthesis
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Standard solvents and reagents were purchased from either Sigma-Aldrich, Chemgenes or Alfa Aesar. 5′-O-(4,4′-Dimethoxytrityl)-5-(3-trifluoroacetylamino-1-propenyl)-2′-deoxyuridine was purchased from Hongene Biotechnology. 0.5 M (1S)-(+)-(10-Camphorsulfonyl)-oxaziridine (CSO) and biotinTEG phosphoramidite were purchased from Glen Research. Sulfo-NHS-acetate was purchased from ThermoFisher Scientific. Thin layer chromatography was carried out on silica gel 60 F254 from either Selecto Scientific (flexible plates) or Fluka (aluminum plates). Flash chromatography was performed on Fluka silica (230–400 mesh). NMR was performed on either a Varian Inova 500 or Bruker 600 MHz instrument. 1H NMR spectra were referenced to the signal of the solvent, and 31P NMR used 2% phosphoric acid as an external reference. FPLC (fast protein liquid chromatography) and HPLC (high performance liquid chromatography) were performed on an AKTA Basic System from GE Healthcare. The protein used in this investigation was human α-thrombin (Haematologic Technologies). The protein was handled according to the manufacturer's recommendations and aliquots were stored at −80°C. The aptamers were stored at −20°C.
10
Purification and Labeling of Anti-hVLDLR Antibody
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