Thirty mg of the melanin granule was dissolved in 10 ml of NaOH (0.1 N) and sonicated for 30 min with a bath type sonicator. Then, 90 mg of mPEG2000-NH2 (Mw = 2,000) and 270 μmol of ethylenediamine were added into the above aqueous solution and stirred with a magnetic stirrer. After vigorous stirring for 12 h, the mixed solution was retrieved by centrifugation (MWCO-10,000, Millipore) at 4,000 rpm for 30 min and washed several times with deionized water. Then, 200 μmol of citraconic anhydride was added into the obtained 10 ml of melanin aqueous solution (1 mg/ml of water) and the pH was adjusted to 9.0 with NaOH (0.1 N). After vigorous stirring for 12 h, mPEG and the citraconic amide modified MNPs were retrieved by centrifugation (MWCO-10,000, Millipore) at 4,000 rpm for 30 min and washed several times with deionized water.
Mwco 10 000
Manufactured by Merck Group
Sourced in United States
The MWCO-10,000 is a molecular weight cut-off filter used for separating and concentrating molecules based on their size. It has a nominal molecular weight limit of 10,000 Daltons, allowing the passage of smaller molecules while retaining larger ones.
Automatically generated - may contain errors
Lab products found in correlation
8 protocols using mwco 10 000
1
Melanin Nanoparticle Synthesis and Functionalization
2
Synthesis of PAMAM-GSH Conjugate
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PAMAM-GSH conjugates were prepared in a six-step sequence. First, PAMAM-SM(PEG)2 was synthesized through the reaction of G4.0 PAMAM dendrimer and SM(PEG)2 in phosphate buffered saline (PBS, pH 7.4) for 2 h at room temperature. The compound obtained from the first step was filtered by ultrafiltration tube (MWCO 10000, Millipore, Bedford, MA Millipore) using ultra centrifuge (Sigma 3K18) to get rid of unreacted SM(PEG)2 and deionized water. Then the product of PAMAM-SM(PEG)2 was resolved in PBS (pH 7.0) and reacted with GSH for 24 h in room temperature. Especially, additional 1% fluorescein was added to the reactive system in order to obtain FITC-Labeled PAMAM-GSH. The final conjugate of PAMAM-GSH was obtained through dialysis by cellulose membranes (MWCO 3000, Union Carbide, NY) for 48 h and lyophilization for 72 h. According to calculation, the substituent degree of GSH in PAMAM-GSH conjugates was 15% theoretically. And the characterization of PAMAM-GSH was conducted by 1H NMR (INOVA, Varian, USA), Fourier transform infrared (FTIR) spectroscopy (NICOLETNexus-470 ARK FTIRUSA).
3
Silk Protein Extraction and Characterization
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Layers of cocoon silks (10 mg) were dissolved in 0.5 mL 9 M LiSCN with vortexing for 2 h. Solubilized proteins were recovered by centrifugation (12,000 g, 10 min, 4°C). Equal amounts of silk proteins (5 μL) were separated on 12.5% (w/v) polyacrylamide gels and visualized by silver nitrite staining. Silk proteins were digested according to the Filter Aided Sample Preparation (FASP) method [15 (link)] and placed in an ultrafiltration tube (MWCO 10,000, Millipore, USA), washed three times with 8 M urea using centrifugation at 12,000 g, 4°C for 20 min, reduced with 15 mM dithiothreitol for 120 min at 37°C and alkylated with 50 mM iodoacetamide for 60 min in the dark. Samples were washed three times with 8 M urea and three times with 50 mM NH4HCO3 and proteins were digested with trypsin at a weight ratio of 1:50 (trypsin:protein) for 20 hours at 37°C. Tryptic peptides were recovered by centrifugation, lyophilized, and resuspended in 80 μL 0.1% formic acid.
4
Melanin Granule Functionalization with mPEG2000-NH2
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Thirty mg of the melanin granule was dissolved in 10 ml of NaOH (0.1 N) and sonicated for 30 min with a bath type sonicator. Then, 90 mg of mPEG2000-NH2 (Mw = 2,000) aqueous solution was dropped into the above aqueous solution and stirred with a magnetic stirrer. After vigorous stirring for 12 h, the mixed solution was retrieved by centrifugation (MWCO-10,000, Millipore) at 4,000 rpm for 30 min and washed several times with deionized water.
5
Dialysis-based DOX Release Kinetics
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The release studies of DOX were carried out by dialysis bag method using phosphate-buffered saline (PBS) as the release medium at pH 7 (corresponding to physiological pH) and pH 5 (to mimic the slightly acidic environment in the tumors). A known amount of DOX-loaded micelles in PBS was placed into a dialysis bag (MWCO 10,000, Sigma-Aldrich), and both ends were sealed. End-sealed dialysis bag was immersed in PBS (pH 5 or 7) shaken at 100 rpm, and temperature was maintained at 37°C. At designated time points, 2 mL of samples was taken from the release medium, and an equal volume of fresh medium was replaced. DOX concentration in the withdrawn sample was determined from the calibration curve using UV absorbance at 482 nm. Drug release curves were obtained by plotting the cumulative % drug release against time.
6
Protein Labeling and Homogeneous Assay
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For labeling of proteins (BSA, phosphorylase B and casein) with His6, the protein was firstly maleoylated with SMPH and secondly labeled with an excess of 9 eq His6-peptide with an N-terminal cysteine side chain (CHHHHHH-NH2). Unreacted linker and peptide were removed by dialysis (MWCO 10,000, Sigma-Aldrich, Taufkirchen, Germany). Protein concentration was determined using absorption at 280 nm. For the homogeneous assay, the “8-4-4” antibody was labeled with BHQ-10 succinimidyl ester. Unreacted dye was removed via SEC on a HiTrap desalting column (5 mL, GE Healthcare, Munich, Germany).
7
Biocompatible Quantum Dot Purification
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The synthesized bio-QDs were collected and resuspended in 10 mM Tris-Cl (pH = 7.6). To isolate the QDs, the resuspended cells were disrupted by high pressure cell disruption device. The crushed cells were then sonication (2 s with 5 s intervals) for 20 times in ice bath. The suspension was centrifuged at 4 000 g for 10 min to collect the fluorescent supernatant. The resulting supernatant was concentrated and washed using a 50 kDa tubular ultrafiltration membrane (MWCO-10000, Merck Millipore Co., USA). To digest the protein impurity, 100 μg/mL proteinase K was added and treated at 37 °C for 1 h. Then resulting solution was purified by centrifugation (15000× g, 10 min) and washing by 50 kDa tubular ultrafiltration membrane and dialysis. Finally, the obtained purified QDs were subjected to high-resolution transmission electron microscopy analysis (TEM-JEM-2010F, JEOL, Tokyo, Japan).
8
Phototropin LOV1 from C. reinhardtii Purification
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Phototropin-LOV1-C57S (CrLOV1) from the unicellular algae C. reinhardtii was expressed from a plasmid carrying an N-terminal 10x His-tag.28 (link) For UV/vis and EPR spectroscopy, CrLOV1 was expressed in Escherichia coli strain BL21 (DE3) using LB-medium. The protein for15N
photo-CIDNP experiments was expressed in M9 minimal medium with 15N isotope-enriched 15NH4Cl.17 (link) Both types are purified by Ni2+-affinity
chromatography (kta purifier, GE Healthcare). Residual imidazole was
removed by dialysis against phosphate buffer (300 mM NaCl and 50 mM
KH2PO4/K2HPO4, pH 8.0).
About 20 mg of protein was concentrated using an ultracentrifugation
filter with a molecular cutoff at 10 kDa (Merck Millipore, MWCO 10000).
The appropriate volume of trehalose solution (1.2 M stock solution)
was added to the concentrated protein sample achieving a molar ratio
of 50:1 (trehalose to protein). The mixture of trehalose and CrLOV1 was dried on a Petri dish under nitrogen gas for
several hours until a solid amorphous glass was formed.
photo-CIDNP experiments was expressed in M9 minimal medium with 15N isotope-enriched 15NH4Cl.17 (link) Both types are purified by Ni2+-affinity
chromatography (kta purifier, GE Healthcare). Residual imidazole was
removed by dialysis against phosphate buffer (300 mM NaCl and 50 mM
KH2PO4/K2HPO4, pH 8.0).
About 20 mg of protein was concentrated using an ultracentrifugation
filter with a molecular cutoff at 10 kDa (Merck Millipore, MWCO 10000).
The appropriate volume of trehalose solution (1.2 M stock solution)
was added to the concentrated protein sample achieving a molar ratio
of 50:1 (trehalose to protein). The mixture of trehalose and CrLOV1 was dried on a Petri dish under nitrogen gas for
several hours until a solid amorphous glass was formed.
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