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Aprotinin

Aprotinin is a potent serine protease inhibitor that plays a crucial role in regulating numerous biological processes.
It is widely used in medical and research applications, including as an antifibrinolytic agent to reduce bleeding during surgery and as a tool for investigating the mechanisms of protease-mediated pathways.
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Most cited protocols related to «Aprotinin»

Molecular Dynamics Simulations of Proteins

Cited 991 times

MD simulations of hen egg white lysozyme (HEWL), bovine pancreatic trypsin inhibitor (BPTI), ubiquitin (Ubq), and the B3 domain of Protein G (GB3) were performed using Desmond version 2.1.0.1 and the Amber ff99SB or the modified Amber ff99SB-ILDN force fields. The TIP3P water model20 was used for simulations of HEWL, Ubq, and GB3, and the TIP4P-Ew water model21 (link) was used for simulations of BPTI. Simulation parameters were the same as in the simulations of small helical peptides, apart from the fact that a 64³ PME grid was used for HEWL and a 48³ grid was used for BPTI, Ubq, and GB3. Simulations of HEWL, BPTI, Ubq, and GB3 were initiated from PDB22 (link) entries 6LYT, 5PTI, 1UBQ, and 1P7E solvated in cubic water boxes containing 10,594, 4215, 6080, and 5156 water molecules, respectively. The net charge of the proteins was neutralized with sodium or chloride ions. Each system was initially subject to energy minimization, followed by 1.2 ns of MD simulation in the NPT ensemble during which the temperature was increased linearly from 10 to 300 K, and position restraints on the backbone atoms were annealed from 1.0 to 0.0 kcal mol⁻¹ Å⁻¹. After this initial relaxation, each system was simulated for 6 ns in the NPT ensemble. The frame of this simulation with the volume closest to the average volume was selected as the starting conformation for a production run of 1.2 μs in the NVT ensemble. The trajectories obtained from the NVT runs were used for subsequent data analysis.

Lindorff-Larsen K., Piana S., Palmo K., Maragakis P., Klepeis J.L., Dror R.O, & Shaw D.E. (2010). Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins, 78(8), 1950-1958.

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Publication 2010

Amber Aprotinin Chlorides Cuboid Bone Helix (Snails) hen egg lysozyme Ions Peptides Protein Domain Proteins Reading Frames Sodium Ubiquitin Vertebral Column

Protein-Protein Interaction Assay

Cited 566 times

Cells were solubilized with lysis buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, 50 mM NaF, 1 mM Na₃VO₄, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF] and centrifuged at 12,000 × g for 20 min. For co-immunoprecipitation assay, cells were sonicated briefly in lysis buffer before centrifugation. The lysates were subjected to immunoprecipitation, SDS-PAGE, and Western blot analyses as described^{2 (link)}.

Nishita M., Park S.Y., Nishio T., Kamizaki K., Wang Z., Tamada K., Takumi T., Hashimoto R., Otani H., Pazour G.J., Hsu V.W, & Minami Y. (2017). Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness. Scientific Reports, 7, 1.

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Publication 2017

Aprotinin Biological Assay Buffers Cells Centrifugation Co-Immunoprecipitation Edetic Acid Immunoprecipitation leupeptin Nonidet P-40 SDS-PAGE Sodium Chloride Tromethamine Western Blot

Molecular Dynamics Simulations of Folded Proteins

Cited 161 times

Simulations using Gromacs 4.5.3 were carried out for the following four folded globular proteins: bovine pancreatic trypsin inhibitor (BPTI), ubiquitin, GB3 and hen Lysozyme, starting from the published experimental structures (PDB entries 5PTI^{48 (link)}, 1UBQ^{49 (link)}, 1P7E^{50 (link)}, 6LYT⁵¹ respectively). Each protein was solvated in a truncated octahedron simulation cell filled with TIP3P water, with nearest distance between images of 45 Å for all proteins except for lysozyme, for which the distance was 60 Å. Sodium and chloride ions were added as needed to yield a final salt concentrations of ~100 mM, with adjustments to ensure charge neutrality. For each protein, a 100-step SD energy minimization of the whole system was performed, followed by 200 ps of MD at a constant pressure of 1 bar and temperature of 300 K, in which harmonic positional restraints of 2.39 kcal/mol/Å² were applied to each Cartesian component of each protein non-hydrogen atom using the minimized structure as a reference. Each protein was then simulated at a constant pressure of 1 bar and a temperature of 300 K for 200 ns. Pressure was regulated by a Parinello-Rahman barostat⁵² with a coupling time of 2.5 ps; otherwise all details were as described for Ac-(AAQAA)₃-NH₂ above.

Best R.B., Zhu X., Shim J., Lopes P.E., Mittal J., Feig M, & MacKerell AD J.r. (2012). Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ1 and χ2 dihedral angles. Journal of chemical theory and computation, 8(9), 3257-3273.

Publication 2012

A 300 Aprotinin Cells Chlorides Eye hen egg lysozyme Hydrogen Ions Muramidase Pressure Proteins Sodium Sodium Chloride Ubiquitin

SaOS2 cells' ER-to-surface transport analysis

Cited 69 times

SaOS2 cells were transfected with the respective siRNAs and then with the respective expression plasmids for VSVG-GFP, VSVG-Myc, VSVG-MT1-MMP, or VSVG-KDELR-Myc with or without expression plasmid for sr-IFT20 or IFT20. To examine the ER-to-cell surface transport, VSVG-GFP-transfected SaOS2 cells were incubated overnight at 40 °C in DMEM containing 1% FBS to accumulate VSVG-GFP at the ER, and then shifted to DMEM containing 1% FBS and 0.1 mg/ml cycloheximide pre-warmed at 32 °C to allow transport through the Golgi. After 30 or 60 min in culture, cell surface proteins were biotinylated with 0.5 mg/ml Sulfo-NHS-ss-biotin (Thermo) in Dulbecco’s PBS (DPBS) for 30 min at 4 °C and quenched with 50 mM NH₄Cl in DPBS for 10 min at 4 °C. After washing with ice-cold DPBS, cells were solubilized with Triton X-100 lysis buffer [25 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.4% (w/v) sodium deoxycholate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF]. Biotinylated proteins were affinity-purified with streptavidin-Sepharose beads and subjected to SDS-PAGE followed Western blot analyses. ER-to-cis-Golgi and intra-Golgi transport of VSVG and VSVG-MT1-MMP and retrograde transport of VSVG-KDELR have been described previously^{48 (link)}.

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Publication 2017

Aprotinin Buffers Cells Cell Surface Proteins Cold Temperature Cycloheximide Deoxycholic Acid, Monosodium Salt Edetic Acid Golgi Apparatus leupeptin Plasmids Proteins RNA, Small Interfering SDS-PAGE Sodium Chloride streptavidin-agarose sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate Triton X-100 Tromethamine Western Blot

Comparative Protein Dynamics by Simulation

Cited 51 times

We simulated four folded proteins for comparison of dynamic properties to NMR. First was the third Igg-binding domain of protein G (GB3). The native structure was defined as a liquid crystal NMR structure (PDB ID: 1P7E^{40 (link)}). Second was the bovine pancreatic trypsin inhibitor (BPTI). The native structure was defined as a joint neutron/X-ray diffraction structure (PDB ID: 5PTI^{41 (link)}). Third was ubiquitin (Ubq), with the native structure defined as a crystal structure (PDB ID: 1UBQ^{42 (link)}). Fourth was hen egg white lysozyme (HEWL), with the native structure defined as a crystal structure (PDB ID: 6LYT⁴³). Owing to their larger size, the proteins were equilibrated as above, but with the unrestrained step extended to a full nanosecond, rather than 500 ps.

Maier J.A., Martinez C., Kasavajhala K., Wickstrom L., Hauser K.E, & Simmerling C. (2015). ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. Journal of chemical theory and computation, 11(8), 3696-3713.

Publication 2015

Aprotinin Binding Proteins hen egg lysozyme Joints Liquid Crystals Protein Domain Proteins Ubiquitin X-Ray Diffraction

Most recents protocols related to «Aprotinin»

Synthesis and Characterization of Porous Chromatography Particles

Not available on PMC !

Example 1

27.8 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 11.3 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were dissolved in 58.7 g of diethyl succinate as a diluent, and nitrogen gas was bubbled for 30 minutes to provide an oil phase.

Next, separately from the oil phase, 10.0 g of PVA-224 (manufactured by Kuraray Co., Ltd., polyvinyl alcohol having a degree of saponification of 87.0% to 89.0%) as a dispersion stabilizer and 10.0 g of sodium chloride as a salting-out agent were dissolved in 480 g of ion exchanged water to provide an aqueous phase.

The aqueous phase and the oil phase were placed in a separable flask and dispersed at a rotation speed of 430 rpm for 20 minutes using a stirring rod equipped with a half-moon stirring blade, then the inside of the reactor was purged with nitrogen, and the reaction was carried out at 60° C. for 16 hours.

After that, the resulting polymer was transferred onto a glass filter and thoroughly washed with hot water at about 50 to 80° C., denatured alcohol, and water in the order presented to obtain 100.4 g of a porous particle (carrier al).

The amount of glycidyl methacrylate used was 79.8 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 20.2 mol % based on the total amount of the monomers.

98 g of the carrier α1 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether. After cleaning, the carrier α1 was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (920 mol % based on glycidyl methacrylate) of 1,4-butanediol were placed in the separable flask, and stirring and dispersion were carried out.

After that, 1.5 ml of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.

The mixture was cooled, then the porous particle (carrier β1) bonded to a diol compound including an alkylene group in the structure thereof was collected by filtration and then washed with 1 L of ion exchanged water to obtain 152 g of a carrier β1.

The progress of the reaction was confirmed by the following procedure.

A part of the dry porous particle into which an alkylene group had been introduced was mixed with potassium bromide, and the resulting mixture was pelletized by applying a pressure and then measured using FT-IR (trade name: Nicolet (registered trademark) iS10, manufactured by Thermo Fisher Scientific Inc.) to check the height of an absorbance peak at 908 cm⁻¹due to the glycidyl group in the infrared absorption spectrum.

As a result, no absorbance peak at 908 cm⁻¹was observed by FT-IR.

150 g of the carrier β1 was weighed onto a glass filter and thoroughly cleaned with dimethylsulfoxide.

After cleaning, the carrier β1 was placed in a separable flask, 262.5 g of dimethyl sulfoxide and 150 g of epichlorohydrin were added, the resulting mixture was stirred at room temperature, 37.5 ml of a 30% sodium hydroxide aqueous solution (manufactured by KANTO CHEMICAL CO., INC.) was further added, and the resulting mixture was heated to 30° C. and stirred for 6 hours.

After completion of the reaction, the obtained product was transferred onto a glass filter and thoroughly washed with water, acetone, and water in the order presented to obtain 172 g of a porous particle into which a glycidyl group had been introduced (carrier γ1).

The introduction density of the glycidyl group in the obtained carrier γ1 was measured by the following procedure.

5.0 g of the carrier γ1 was sampled, and the dry mass thereof was measured and as a result, found to be 1.47 g. Next, the same amount of the carrier γ1 was weighed into a separable flask and dispersed in 40 g of water, 16 mL of diethylamine was added while stirring at room temperature, and the resulting mixture was heated to 50° C. and stirred for 4 hours. After completion of the reaction, the reaction product was transferred onto a glass filter and thoroughly washed with water to obtain a porous particle A into which diethylamine had been introduced.

The obtained porous particle A was transferred into a beaker and dispersed in 150 mL of a 0.5 mol/L potassium chloride aqueous solution, and titration was carried out using 0.1 mol/L hydrochloric acid with the point at which the pH reached 4.0 as the neutralization point.

From this, the amount of diethylamine introduced into the porous particle A into which diethylamine had been introduced was calculated, and the density of the glycidyl group of the carrier γ1 was calculated from the following expression.

As a result, the density of the glycidyl group was 880 μmol/g.
Density(μmol/g) of glycidyl group={0.1×volume(μL) of hydrochloric acid at neutralization point/dry mass(g) of porous particle into which glycidyl group has been introduced}<Step (D): Introduction Reaction of Polyol>

150 g of the carrier γ1, 600 mL of water, and 1000 g (13000 mol % based on glycidyl group) of D-sorbitol (log P=−2.20, manufactured by KANTO CHEMICAL CO., INC.) were placed in a 3 L separable flask and stirred to form a dispersion.

After that, 10 g of potassium hydroxide was added, the temperature was raised to 60° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 15 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a porous particle into which polyol had been introduced (carrier 61).

The obtained carrier 61 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 1.

The alkali resistance was evaluated by calculating the amount of a carboxy group produced by hydrolysis of sodium hydroxide according to the following procedure.

First, 4 g of the packing material was dispersed in 150 mL of a 0.5 mol/L potassium chloride aqueous solution, and titration was carried out using 0.1 mol/L sodium hydroxide aqueous solution with the point at which the pH reached 7.0 as the neutralization point. From this, the amount of a carboxy group before hydrolysis included in the packing material was calculated from the following expression.
Amount(μmol/mL) of carboxy group=0.1×volume(μL) of sodium hydroxide aqueous solution at the time of neutralization/apparent volume (mL) of packing material

Here, the apparent volume of the packing material is the volume of the packing material phase measured after preparing a slurry liquid by dispersing 4 g of the packing material in water, transferring the slurry liquid to a graduated cylinder, and then allowing the same to stand for a sufficient time.

Subsequently, 4 g of the packing material was weighed into a separable flask, 20 mL of a 5 mol/L sodium hydroxide aqueous solution was added, and the resulting mixture was treated at 50° C. for 20 hours while stirring at 200 rpm. The mixture was cooled, then the packing material was collected by filtration, then washed with a 0.1 mol/L HCl aqueous solution and water in the order presented, and the amount of a carboxy group contained in the obtained packing material was calculated by the same method as above. From the difference between the amount of a carboxy group before and that after the reaction with the 5 mol/L sodium hydroxide aqueous solution, the amount of a carboxy group produced by the reaction with the 5 mol/L sodium hydroxide aqueous solution was calculated. As a result, the amount of a carboxy group produced was 21 μmol/mL.

If the amount of a carboxy group produced is 40 μmol/mL or less, the alkali resistance is considered to be high.

The obtained packing material was packed into a stainless steel column (manufactured by Sugiyama Shoji Co., Ltd.) having an inner diameter of 8 mm and a length of 300 mm by a balanced slurry method. Using the obtained column, a non-specific adsorption test was carried out by the method shown below.

The column packed with the packing material was connected to a Shimadzu Corporation HPLC system (liquid feed pump (trade name: LC-10AT, manufactured by Shimadzu Corporation), autosampler (trade name: SIL-10AF, manufactured by Shimadzu Corporation), and photodiode array detector (trade name: SPD-M10A, manufactured by Shimadzu Corporation)), and a 50 mmol/L sodium phosphate buffer aqueous solution as a mobile phase was passed at a flow rate of 0.6 mL/min.

Using the same sodium phosphate aqueous solution as the mobile phase as a solvent, their respective sample solutions of 0.7 mg/mL thyroglobulin (Mw of 6.7×105), 0.6 mg/mL γ-globulin (Mw of 1.6×105), 0.96 mg/mL BSA (Mw of 6.65×104), 0.7 mg/mL ribonuclease (Mw of 1.3×104), 0.4 mg/mL aprotinin (Mw of 6.5×103), and 0.02 mg/mL uridine (Mw of 244) (all manufactured by Merck Sigma-Aldrich) are prepared, and 10 μL of each is injected from the autosampler.

The elution time of each observed using the photodiode array detector at a wavelength of 280 nm was compared to confirm that there was no contradiction between the order of elution volume and the order of molecular weight size.

As a result, the elution volumes of the samples from the column packed with the packing material 1 were 8.713 mL, 9.691 mL, 9.743 mL, 10.396 mL, 11.053 mL, and 11.645 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced. When there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof, there was no non-specific adsorption, which is indicated as 0 in Table 1, and when there was a contradiction therebetween, non-specific adsorption was induced, which is thus indicated as X.

The porous particle (carrier al) obtained in the same manner as in Example 1 was subjected to the step D of Example 1.

98 g of carrier al, 600 mL of water, and 1000 g (3050 mol % based on glycidyl group) of D-sorbitol (manufactured by KANTO CHEMICAL CO., INC.) were placed in a 3 L separable flask and stirred to form a dispersion.

After that, 10 g of potassium hydroxide was added, the temperature was raised to 60° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 15 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 130 g of a porous particle into which a polyol had been introduced (carrier δ7).

The carrier δ7 was classified into 16 to 37 μm using a sieve to obtain 115 g of a packing material 7.

The alkali resistance of the obtained packing material 7 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced in the packing material 7 was 120.3 μmol/mL, resulting in poor alkali resistance.

Further, the non-specific adsorption of the obtained packing material 7 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.606 mL, 9.769 mL, 9.9567 mL, 10.703 mL, 11.470 mL, and 12.112 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

Example 2

A porous particle (carrier al) was obtained in the same manner as in Example 1, and then a packing material 2 was obtained as follows.

98 g of the carrier α1 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether.

After cleaning, the porous particle was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (580 mol % based on the glycidyl group) of 1,4-cyclohexanedimethanol were placed in the separable flask, and stirring and dispersion were carried out.

The mixture was cooled, then the resulting porous particle (carrier $2) bonded to a diol compound including an alkylene group in the structure thereof was collected by filtration and then washed with 1 L of ion exchanged water to obtain 165 g of a carrier 32.

The progress of the reaction was confirmed by the following procedure.

A part of the dry porous particle into which an alkylene group had been introduced was mixed with potassium bromide, and the resulting mixture was pelletized by applying a pressure and then measured using FT-IR (trade name: Nicolet (registered trademark) iS10, manufactured by Thermo Fisher Scientific Inc.) to check the height of a absorbance peak at 908 cm⁻¹due to the glycidyl group in the infrared absorption spectrum.

As a result, no absorbance peak at 908 cm⁻¹was observed by FT-IR.

150 g of the carrier $2 was weighed onto a glass filter and thoroughly cleaned with dimethylsulfoxide. After cleaning, the carrier $2 was placed in a separable flask, 262.5 g of dimethyl sulfoxide and 150 g of epichlorohydrin were added, the resulting mixture was stirred at room temperature, 37.5 ml of a 30% sodium hydroxide aqueous solution (manufactured by KANTO CHEMICAL CO., INC.) was further added, and the resulting mixture was heated to 30° C. and stirred for 6 hours. After completion of the reaction, the porous particle was transferred onto a glass filter and thoroughly washed with water, acetone, and water in the order presented to obtain 180 g of a porous particle into which a glycidyl group had been introduced (carrier γ2).

The introduction density of the glycidyl group in the obtained carrier γ2 was measured in the same manner as in Example 1. As a result, the density of the glycidyl group was 900 μmol/g.

150 g of the carrier γ2 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether. After cleaning, the carrier γ2 was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (5760 mol % based on the glycidyl group) of ethylene glycol (log P=−1.36) were placed in the separable flask, and stirring and dispersion were carried out. After that, 1.5 mL of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours. The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a polyol-introduced porous particle (carrier δ2). The carrier δ2 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 2.

The alkali resistance of the obtained packing material 2 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 15.2 μmol/mL, and it was confirmed that the packing material 2 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 2 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.814 mL, 9.635 mL, 9.778 mL, 10.37 mL, 10.898 mL, and 12.347 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 8 was obtained in the same manner as in Example 1 except that 150 g of ethylene glycol was used instead of 1,4-butanediol as an alkylene group-introducing agent.

The alkali resistance of the obtained packing material 8 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced in the packing material 8 was 108.4 μmol/mL, resulting in poor alkali resistance.

Further, the non-specific adsorption of the obtained packing material 8 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.708 mL, 9.8946 mL, 10.6452 mL, 11.5374 mL, and 12.1656 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

Example 3

A carrier γ2 was obtained in the same manner as in Example 2.

150 g of the obtained carrier γ2 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether.

After cleaning, the porous particle was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g of polyethylene glycol #200 (manufactured by KANTO CHEMICAL CO., INC., average molecular weight of 190 to 210, log P is unclear, but the close compound tetraethylene glycol (Mw of 194) has a log P of −2.02) (1790 mol % based on glycidyl group) were placed in the separable flask, and stirring and dispersion were carried out.

After that, 1.5 mL of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a porous particle into which a polyol had been introduced (carrier 63).

The carrier δ3 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 3.

The alkali resistance of the obtained packing material 3 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 16.1 μmol/mL, and it was confirmed that the packing material 3 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 3 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.517 mL, 9.241 mL, 9.47 mL, 10.034 mL, 10.484 mL, and 11.927 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 9 was obtained in the same manner as in Example 2 except that no glycidyl group was introduced and no polyol was introduced. That is, the carrier $2 obtained in the step (B) of Example 2 was used as the packing material 9.

The non-specific adsorption of the obtained packing material 9 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.590 mL, 10.316 mL, 9.603 mL, 10.484 mL, 13.863 mL, and 12.861 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

Example 4

A packing material 4 was obtained in the same manner as in Example 3 except that 33.2 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 5.9 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 90.0 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 10.0 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 4 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 11.5 μmol/mL, and it was confirmed that the packing material 4 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 4 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 7.52 mL, 8.214 mL, 8.451 mL, 9.062 mL, 9.511 mL, and 11.915 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 10 was obtained in the same manner as in Example 1 except that 150 g (480 mol % based on glycidyl methacrylate) of 1,10-decanediol was used instead of 1,4-butanediol as an alkylene group-introducing agent.

The non-specific adsorption of the obtained packing material 10 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.991 mL, 10.15 mL, 10.063 mL, 10.691 mL, 12.172 mL, and 11.531 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

Example 5

A packing material 5 was obtained in the same manner as in Example 3 except that 21.5 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 17.6 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase.

The amount of glycidyl methacrylate used was 66.2 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 33.8 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 5 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 18.3 μmol/mL, and it was confirmed that the packing material 5 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 5 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.692 mL, 9.434 mL, 9.625 mL, 10.236 mL, 10.759 mL, and 12.457 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 11 was obtained in the same manner as in Example 3 except that 13.7 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 25.4 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 46.4 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 53.6 mol % based on the total amount of the monomers.

The non-specific adsorption of the obtained packing material 11 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.872 mL, 10.131 mL, 9.82 mL, 10.422 mL, 12.782 mL, and 12.553 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

It was confirmed that the exclusion limit molecular weights of the packing materials obtained in Examples 1 to 6 and Comparative Examples 1 to 5 were all 1,000,000 or more.

Example 6

A packing material 6 was obtained in the same manner as in Example 3 except that 33.2 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 5.9 g of ethylene glycol dimethacrylate (trade name: NK Ester 1G, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 29.3 g of butyl acetate, 29.3 g of chlorobenzene, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 88.7 mol % based on the total amount of the monomers, and the amount of ethylene glycol dimethacrylate used was 11.3 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 6 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 12.5 μmol/mL, and it was confirmed that the packing material 6 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 6 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.613 mL, 10.427 mL, 10.444 mL, 11.066 mL, 11.582 mL, and 12.575 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 12 was obtained in the same manner as in Example 3 except that 37.1 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 2.0 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 96.7 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 3.3 mol % based on the total amount of the monomers.

Packing into a stainless steel column using the obtained packing material 12 was attempted. However, the back pressure was high, making liquid feeding difficult, and this made it impossible to carry out the packing. Because of this, neither of the evaluations was able to be carried out.

Results of the above Examples and Comparative Examples are shown in Table 1.

From the above results, by adopting the configuration of the present invention, a packing material having suppressed non-specific adsorption and high alkali resistance can be obtained.

When no hydrophobic portion is provided or when the alkylene chain is short, the alkali resistance is low as shown in Comparative Examples 1 and 2. In addition, it was found that when the alkylene chain is too long or when no hydrophilic portion is provided, the hydrophobicity is strong, and non-specific adsorption is induced as shown in Comparative Examples 3 and 4. In addition, in Comparative Example 5 having many repeating units derived from a polyfunctional monomer, it was found that non-specific adsorption was induced, and in Comparative Example 6 having fewer repeating units derived from a polyfunctional monomer, it was found that the back pressure applied to the apparatus was high, making column packing difficult.

TABLE 1

Amount of

carboxy

Degree ofgroup

PolyfunctionalcrosslinkingNon-specificproduced

Monomer[mol %]Alkylene groupPolyoladsorption⁵⁾[μmol/mL]

Ex. 1GDMA¹⁾20.2Butylene groupSorbitol◯21

Ex. 2GDMA20.2Cyclohexane-1,4-dimethyleneEG³⁾◯15.2

group

Ex. 3GDMA20.2Cyclohexane-1,4-dimethylenePEG200⁴⁾◯16.1

group

Ex. 4GDMA10Cyclohexane-1,4-dimethylenePEG200◯11.5

group

Ex. 5GDMA33.8Cyclohexane-1,4-dimethylenePEG200◯18.3

group

Ex. 6EDMA²⁾11.3Cyclohexane-1,4-dimethylenePEG200◯12.5

group

Comp.GDMA20.2—Sorbitol◯120.3

Ex. 1

Comp.GDMA20.2Ethylene groupEG◯108.4

Ex. 2

Comp.GDMA20.2Cyclohexane-1,4-dimethylene—X—

Ex. 3group

Comp.GDMA20.2Decanylene groupSorbitolX—

Ex. 4

Comp.GDMA53.6Cyclohexane-1,4-dimethylenePEG200X—

Ex. 5group

Comp.GDMA3.3Cyclohexane-1,4-dimethylenePEG200Unmeasurable

Ex. 6group

¹⁾GDMA: Glycerin-1,3-dimethacrylate

²⁾EDMA: Ethylene glycol dimethacrylate

³⁾EG: Ethylene glycol

⁴⁾PEG200: Polyethylene glycol #200

⁵⁾◯: No non-specific adsorption, X: Non-specific adsorption

US11857949B2. Packing material and method for producing the same, and column for size exclusion chromatography (2024-01-02). Resonac Corporation [JP]. Inventors: Naoki Uchiyama [JP], Yuzuru Kokido [JP].

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Patent 2024

A 300 Acetone Adsorption Alkalies Anabolism Aprotinin boron trifluoride Buffers butyl acetate butylene Butylene Glycols chlorobenzene COMP protocol Cyclohexane cyclohexanedimethanol diethylamine diethyl succinate diglyme Epichlorohydrin Esters Ethanol ethylene dimethacrylate Ethylenes Ethyl Ether Filtration G 130 gamma-Globulin Gel Chromatography Glycerin glycidyl methacrylate Glycol, Ethylene High-Performance Liquid Chromatographies Hydrochloric acid Hydrolysis Nitrogen Polyethylene Glycols Polymers polyol Polyvinyl Alcohol potassium bromide Potassium Chloride potassium hydroxide Pressure Ribonucleases Sodium Hydroxide sodium phosphate Solvents Sorbitol Stainless Steel Sulfoxide, Dimethyl tetraethylene glycol Thyroglobulin Titrimetry Uridine

Optimizing Protease Inhibitor Cocktail for Buccal Samples

Not available on PMC !

Example 5

Optimizing Inhibitor Cocktail to Maximize Buccal Sample Non-Specific Protease Activity Inhibition

29 inhibitors were tested using a competitive enzymatic activity assay of 3CL protease in buccal samples.

PMSF, GW, aprotinin, eglinC and pepstatin all decreased non-specific protease activity in saliva, with a non significant inhibition of 3CL specific activity.

The four best inhibitors were Eglin C, GW, PMSF and 2,6 PDA.

Various inhibitor cocktails were also tested:

- Cocktail 1 (PMSF, GW);
- Cocktail 2 (Eglin C, GW, PMSF and 2,6 PDA)
- Cocktail 2 reduced non-specific proteases to a greater degree than cocktail 1.

US11879893B2. Rapid detection test for SARS-CoV-2 (2024-01-23). NLC Pharma Ltd [IL]. Inventors: Dorit Arad [IL].

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Patent 2024

Aprotinin eglin C Endopeptidases Enzyme Assays inhibitors pepstatin Peptide Hydrolases Protease Inhibitors Psychological Inhibition SARS-CoV-2 Xerostomia

Western Blot Protein Expression Analysis

Protein lysates were prepared in RIPA-buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 1 mM sodium dodecyl sulfate, 10 μg/mL aprotinin, 1 mM phenylmethanesulfonyl fluoride, and 10 μg/mL leupeptin) supplemented with complete protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentration was determined using the BCA protein assay kit (ThermoFisher Scientific, Carlsbad, CA, USA). Cell lysate (50 μg) was separated by electrophoresis on SDS-PAGE gel and transferred to nitrocellulose membranes (Pall Corporation, Washington, NY, USA). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and infiltrated overnight at 4 °C with specific antibodies. Antibodies used in this study were listed in the Supplementary Information. Membranes were washed three times in PBST the next day, then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, washed three times with PBST and finally bands were visualized using an enhanced chemiluminescence system (Millipore, Los Angeles, CA USA). The representative western blot images for at least three independent experiments shown in the figures have been cropped and auto contrasted.

Li X., Wang S., Xie Y., Jiang H., Guo J., Wang Y., Peng Z., Hu M., Wang M., Wang J., Li Q., Wang Y, & Liu Z. (2023). Deacetylation induced nuclear condensation of HP1γ promotes multiple myeloma drug resistance. Nature Communications, 14, 1290.

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Publication 2023

Antibodies Aprotinin Biological Assay Buffers Cells Chemiluminescence Deoxycholic Acid, Monosodium Salt Edetic Acid Electrophoresis Horseradish Peroxidase leupeptin Milk, Cow's Nitrocellulose Nonidet P-40 Phenylmethylsulfonyl Fluoride Protease Inhibitors Proteins Radioimmunoprecipitation Assay SDS-PAGE Sodium Chloride Sulfate, Sodium Dodecyl Tissue, Membrane Tromethamine Western Blotting

Immunoprecipitation of Neural Tubes

Neural tubes were collected from ICR mouse E9.5 embryos and lysed in PBS containing 1% NP-40 and 10 μg/ml aprotinin. Immunoprecipitation and western blotting were performed as previously described^{44 (link)}.

Nagaoka T., Katsuno T., Fujimura K., Tsuchida K, & Kishi M. (2023). Functional interaction between Vangl2 and N-cadherin regulates planar cell polarization of the developing neural tube and cochlear sensory epithelium. Scientific Reports, 13, 3905.

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Publication 2023

Aprotinin Embryo Immunoprecipitation Mice, Inbred ICR Nonidet P-40 Tube, Neural

Phospho-Mypt1 Detection in Neural Tubes

Neural tubes were collected from E9.5 embryos and lysed in PBS containing 1% NP-40, PhosSTOP (Roche, Mannheim, Germany), and 10 μg/ml aprotinin prior to analysis by western blotting. Chemiluminescent signal intensity was measured using the Fiji gel analyser tool. The signal intensity of phospho-Mypt1 was standardized to the signal intensity of Mypt1.

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Publication 2023

Aprotinin Embryo Nonidet P-40 Tube, Neural

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Aprotinin by Merck Group

Sourced in United States, Germany, United Kingdom, Italy, China, Canada, Japan, Spain, France, Israel, Belgium, Austria, Switzerland, Finland, India, Australia, Macao, Hungary, Sweden, Sao Tome and Principe

Aprotinin is a protease inhibitor derived from bovine lung tissue. It is used as a laboratory reagent to inhibit protease activity in various experimental procedures.

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PVDF membranes are a type of laboratory equipment used for a variety of applications. They are made from polyvinylidene fluoride (PVDF), a durable and chemically resistant material. PVDF membranes are known for their high mechanical strength, thermal stability, and resistance to a wide range of chemicals. They are commonly used in various filtration, separation, and analysis processes in scientific and research settings.

Phenyl methyl sulfonyl fluoride (pmsf) by Merck Group

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Bradford assay by Bio-Rad

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Bca protein assay kit by Thermo Fisher Scientific

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Protein assay by Bio-Rad

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The Bio-Rad protein assay is a colorimetric detection and quantitation method for measuring the total protein content in a sample. It utilizes a dye-binding reagent that changes color when bound to proteins, allowing for the determination of protein concentration through spectrophotometric analysis.

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Nitrocellulose membranes are a type of laboratory equipment designed for use in protein detection and analysis techniques. These membranes serve as a support matrix for the immobilization of proteins, enabling various downstream applications such as Western blotting, dot blotting, and immunodetection.

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β actin by Merck Group

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What are the key features and benefits of using Aprotinin?

Aprotinin is a potent serine protease inhibitor that plays a crucial role in regulating numerous biological processes. It is widely used in medical and research applications due to its ability to reduce bleeding during surgery as an antifibrinolytic agent, as well as its utility as a tool for investigating the mechanisms of protease-mediated pathways. Aprotinin helps maintain the delicate balance of proteolytic activity, which is essential for proper physiological function.

What are the different variations or types of Aprotinin available?

Aprotinin is available in various forms, including purified from bovine lung, recombinant, and synthetic versions. Each type may have slightly different properties and applications, so it's important to carefully select the appropriate Aprotinin for your specific research needs. PubCompare.ai can help you identify the optimal Aprotinin product by comparing the characteristics and performance of different Aprotinin variants.

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What are some common challenges or considerations when using Aprotinin?

One key consideration when using Aprotinin is ensuring proper dosage and administration. Overuse of Aprotinin can lead to potential side effects, such as an increased risk of thrombotic events. Additionally, some research has suggested that Aprotinin may interfere with certain diagnostic tests, so it's important to be aware of these potential limitations. PubCompare.ai can assist in navigating these challenges by providing detailed comparisons of Aprotinin protocols and products, helping researchers make informed decisions about its use.

More about "Aprotinin"

Aprotinin, a potent serine protease inhibitor, plays a crucial role in regulating numerous biological processes.
It is widely used in medical and research applications, such as an antifibrinolytic agent to reduce bleeding during surgery and as a tool for investigating the mechanisms of protease-mediated pathways.
Researchers can optimize their Aprotinin studies by leveraging PubCompare.ai's AI-powered platform.
This tool enables quick identification of the best protocols and products from literature, preprints, and patents, enhancing the reproducibility and accuracy of Aprotinin experiments.
When investigating Aprotinin-related pathways, researchers may also utilize other protease inhibitors like Leupeptin, as well as protein quantification techniques such as the Bradford assay, BCA protein assay kit, and Bio-Rad protein assay.
Additionally, Western blotting methods employing PVDF or nitrocellulose membranes, and the detection of housekeeping proteins like β-actin, can provide valuable insights.
By leveraging these comprehensive resources and techniques, scientists can confidently advance their Aprotinin-focused studies and make meaningful discoveries in their field of research.