Hydrophobic polymers including poly(lactic-co-glycolic acid) (PLGA, 50:50 lactic:glycolic acid, MW 30,000–60,000), poly(L-lactic acid) (PLLA, MW 50,000), and polycaprolactone (PCL, MW 80,000), and the hydrophilic polymer, polyethylene oxide (PEO, MW 100,000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris-EDTA (TE) buffer (pH 8.0), phosphate buffered saline (PBS), and the organic solvents chloroform, dimethyl sulfoxide (DMSO), and hexafluoroisopropanol (HFIP) were also purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were used directly without further purification. One milliliter plastic syringes, petri dishes, and 20 mL scintillation vials were obtained from VWR. One milliliter glass syringes were purchased from Fisher Scientific. The electrospinner was provided courtesy of Dr. Stuart Williams at the Cardiovascular Innovative Institute, University of Louisville.
Hexafluoroisopropanol hfip
Manufactured by Merck Group
Sourced in United States
Hexafluoroisopropanol (HFIP) is a specialty chemical compound used primarily as a solvent and reagent in various laboratory applications. It has a unique chemical structure with a high degree of fluorination, which gives it distinct physical and chemical properties. HFIP is known for its ability to dissolve a wide range of organic compounds, making it a versatile tool in chemical synthesis and analysis.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Thioflavin t tht, by Merck Group (2 mentions)
Trifluoroacetic acid tfa, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Scintillation vials, by Avantor (1 mentions)
Polycaprolactone pcl, by Merck Group (1 mentions)
Petri dishes, by Avantor (1 mentions)
Polyethylene oxide peo, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Ultrapure water, by Sartorius (1 mentions)
Ammonium acetate solution, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Carbenicillin, by GoldBio (1 mentions)
Boric acid, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol, by GoldBio (1 mentions)
Lysozyme, by GoldBio (1 mentions)
Dpn1 enzyme, by New England Biolabs (1 mentions)
Saccharomyces cerevisiae trnaphe, by Merck Group (1 mentions)
Isopropyl β d thiogalactoside, by GoldBio (1 mentions)
Tris cl, by Thermo Fisher Scientific (1 mentions)
15 protocols using hexafluoroisopropanol hfip
1
Fabrication of Electrospun Polymer Scaffolds
2
Modulation of Antimicrobial Peptide Structure
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PSM native peptides and shorter derivatives (custom synthesis) at >98% purity (see Table 2 ) were purchased from GL Biochem (Shanghai) Ltd., as well as from GenScript. The short (6–7 residues) PSMα segments were synthesized with capped termini (acetylated in the N-terminus and amidated in the C-terminus) or with unmodified termini for crystallography. PSMα full-length peptides were synthesized with unmodified termini. Hexafluoroisopropanol (HFIP) and Thioflavin T (ThT) were purchased from Sigma-Aldrich. Ultra-pure water was purchased from Biological Industries. Dimethyl-sulfoxide (DMSO) was purchased from Merck.
PSMαs wild type (WT) and mutant peptide sequences (Uniprot accession codes are in parenthesis)
| Peptide name | Sequence |
|---|---|
| PSM α1 (H9BRQ5) | MGIIAG |
| PSM α1 mutant I7P/K9P | MGIIAG |
| PSM α3 (H9BRQ7) | MEFVAK |
| PSM α4 (H9BRQ8) | MAIVGT |
| PSM α4 mutant I8P/I10P | MAIVGTI |
Segments of short derivatives used here are marked in bold and introduced proline substitutions are underlined
3
Site-Directed Mutagenesis Protocol for Protein Engineering
4
In vitro Inhibition of Amyloid-Beta Oligomerization
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For in vitro inhibition tests of oligomerization/ fibrillation, we used lyophilized amyloid-β 1–40 (Aβ40) and lyophilized Methionine amyloid-β 1–40 (MAβ40) [33 (link)] which were bought from rPeptide LLC (Bogart, GA, USA).
For sample preparation, the first weighing of amyloid-β (Aβ) with a high precision balance was completed, then Hexafluoroisopropanol (HFIP; Sigma Aldrich Company) was added to achieve 5–20 μM solutions for concentration adjustments related to pre-measurements of the peptide quality. Furthermore, such treated Aβ were pipetted into marked tubes, vacuum evaporated for 24 h and finally frozen at –80°C. These samples were stored at the same temperature for a minimum of one day but less than two weeks until usage [34–37 (link)].
For sample preparation, the first weighing of amyloid-β (Aβ) with a high precision balance was completed, then Hexafluoroisopropanol (HFIP; Sigma Aldrich Company) was added to achieve 5–20 μM solutions for concentration adjustments related to pre-measurements of the peptide quality. Furthermore, such treated Aβ were pipetted into marked tubes, vacuum evaporated for 24 h and finally frozen at –80°C. These samples were stored at the same temperature for a minimum of one day but less than two weeks until usage [34–37 (link)].
5
Synthesis and Characterization of AB/CO2 Polymer
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CS with a low viscosity was purchased from the Aladdin Co. (Los Angeles, CA, USA). AB (99.8% purity) was purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), and CO2 of 99.9% purity was purchased from the Rihong Air Products Co., Ltd. (Xiamen, China). PVM/MA (analytical purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hexafluoroisopropanol (HFIP, analytical purity) was purchased from Sigma-Aldrich (USA).
6
Amyloid Peptide Preparation and Characterization
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The peptides Aβ40, Aβ42, and Aβ43 were purchased from Peptides International (Peptide Institute, Inc., Osaka, Japan). The peptides were TFA salts. Hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) were purchased from Sigma (St. Louis, MO) and Sigma-Aldrich (St. Louis, MO), respectively. Identities of the peptides were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
7
PLGA Biomaterial for Tissue Engineering
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PLGA (LA : GA = 75 : 25, molecular weight 130 000, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin Province, China), hexafluoroisopropanol (HFIP) (analytically pure, Sigma-Aldrich, St. Louis, MO, USA), dopamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA), bFGF (UB Biotech, Buffalo, NY, USA), ponericin G1 peptide (Top-peptide, Shanghai, China), Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (Life Technologies, Carlsbad, CA, USA), penicillin (Huabei Pharmaceutical Factory, Shijiazhuang, China), streptomycin (North China Pharmaceutical Factory, Shijiazhuang, China), trypsin (Solarbio, Beijing, China), and methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Gibco, Gaithersburg, MD, USA) were acquired from various sources. Phosphate-buffered saline (PBS) was prepared in-house. Fluorescein isothiocyanate (FITC) and 4′,6-bis(2′-imidazolinyl-4H,5H)-2-phenylindole (DIPI) were purchased from Sigma-Aldrich.
Equipment included a field emission scanning electron microscope (ES EM, XL30ESEM-FEG, FEL, Netherlands), fluorescence inverted microscope (Eclipse TE2000-U, Nikon, Tokyo, Japan), carbon dioxide incubator (Thermo Fisher Scientific, Waltham, MA, USA), contact angle tester (Theta Lite, KSV Instrument, Finland), and universal testing machine (Instron, Norwood, MA, USA).
Equipment included a field emission scanning electron microscope (ES EM, XL30ESEM-FEG, FEL, Netherlands), fluorescence inverted microscope (Eclipse TE2000-U, Nikon, Tokyo, Japan), carbon dioxide incubator (Thermo Fisher Scientific, Waltham, MA, USA), contact angle tester (Theta Lite, KSV Instrument, Finland), and universal testing machine (Instron, Norwood, MA, USA).
8
Neuroprotective Potential of Dolichos falcata
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The plant roots of Dolichos falcata Klein were bought from medicinal market in Yunnan province, China. The herbs were identified by Prof. Dingrong Wang, College of Pharmaceutical Sciences, South-Central University for Nationalities, China. Aβ42 peptide was purchased from GL Biochem (Shanghai) Ltd. (Shanghai, China) with the purity of higher than 95%. The amino acid sequence of Aβ42 is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA. Aβ42 was kept in –80°C fringe. Hexafluoroisopropanol (HFIP) with the purity of more than 99.5% was purchased from Sigma (St. Louis, MO, USA). Methanol (HPLC gradient grade) and butanol (AR grade) were purchase from China National Pharmaceutical Group Co. (Beijing, China). Other chemicals and agents were all of the analytic grades and bought from local markets in Beijing, China.
9
Synthesis and Characterization of Staphylococcal Peptides
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Peptide sequences:
PSMα1:fMGIIAGIIKVIKSLIEQFTGK
PSMα2:fMGIIAGIIKFIKGLIEKFTGK
PSMα3:fMEFVAKLFKFFKDLLGKFLGNN
PSMα4:fMAIVGTIIKIIKAIIDIFAK
fδ-toxin: fMAQDIISTISDLVKWIIDTVNKFTKK
dfδ-toxin: MAQDIISTISDLVKWIIDTVNKFTKK
The peptides were synthesized according to the previous report [11 (link)] by Sangon Biotech. The purity of the peptides was > 95%. Trifluoroacetic acid (TFA), hexafluoroisopropanol (HFIP), and Thioflavin T (ThT) were purchased from Sigma-Aldrich. Bis(sulfosuccinimidyl) suberate (BS3), and dithiothreitol (DTT) were purchased from Thermo Fisher Scientific. Ultra-pure water was purchased from Millipore Sigma.
PSMα1:fMGIIAGIIKVIKSLIEQFTGK
PSMα2:fMGIIAGIIKFIKGLIEKFTGK
PSMα3:fMEFVAKLFKFFKDLLGKFLGNN
PSMα4:fMAIVGTIIKIIKAIIDIFAK
fδ-toxin: fMAQDIISTISDLVKWIIDTVNKFTKK
dfδ-toxin: MAQDIISTISDLVKWIIDTVNKFTKK
The peptides were synthesized according to the previous report [11 (link)] by Sangon Biotech. The purity of the peptides was > 95%. Trifluoroacetic acid (TFA), hexafluoroisopropanol (HFIP), and Thioflavin T (ThT) were purchased from Sigma-Aldrich. Bis(sulfosuccinimidyl) suberate (BS3), and dithiothreitol (DTT) were purchased from Thermo Fisher Scientific. Ultra-pure water was purchased from Millipore Sigma.
10
Characterization of SFV21 Peptide Interactions
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Lipids (dimirystoyl-phosphatidylcholine (DMPC), dimirystoyl-phosphatidylglycerol (DMPG), dimirystoyl-phosphatidylethanolamine (DMPE) and cholesterol) were purchased from Sigma. SFV21 peptide: YQCKVYTGVYPFMWGGAYCFC was purchased from GenScript (Piscataway, USA). SFV21 was solubilized in hexafluoroisopropanol (HFIP) from Sigma at micromolar concentrations. Peptide concentrations were calculated from the absorbance at 280 nm using a molar extinction coefficient ε280 of 11,200 M− 1·cm− 1. The molar extinction coefficient of the peptide has been determined using the formula ε 280nm = 5600 × nW + 1400 × nY which takes into account the individual extinction coefficient of the amino acids W and Y. A Raman spectrum of a dried film of SFV21 after incubation in buffer (Supplementary data S1) was performed to insure that the three cysteins in the sequence remain non oxydized in the experimental conditions used (no band characteristic of S-S bond around 520–540 cm− 1, only a band characteristic of S—H bond in the 2500–2600 cm− 1 region).
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