All fluorescent protein coding sequences were inserted between BamHI and EcoRI sites in the constitutive expression vector pNCS which encodes an N-terminal 6×His tag and linker. Sequences for all primers used in this study are listed in Supplementary Table 4 . Fluorescent proteins were expressed in E. coli strain NEBTurbo (New England Biolabs) or Mach1 (Invitrogen) by growing cultures in 2×YT medium supplemented with ampicillin overnight at 37 °C and shaking at 250 rpm. Fluorescent proteins were purified by Ni2+-affinity chromatography as previously described9 (link). Proteins were eluted in 50 mM Tris pH 7.5 or 50 mM sodium phosphate buffer pH 7.5 containing 250 mM imidazole. For all further characterization experiments, eluted fluorescent proteins were buffer-exchanged using Amicon Ultra0.5 10 kD MWCO ultrafiltration units (Millipore) into the same buffer without imidazole. Proteins were found to be stable when stored at 4°C indefinitely or when frozen at −20 °C or −80 °C.
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Therapeutic or Preventive Procedure
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Ultrafiltration
Ultrafiltration
Ultrafiltration is a separation technique used to purify and concentrate solutes in a solution.
It involves passing the solution through a semipermeable membrane, allowing smaller molecules and solvents to pass through while retaining larger molecules and particles.
This process is commonly used in biomedical research, pharmaceutical development, and water treatment to isolate and concentrate biomolecules, proteins, and other macromolecules.
Ultrafiltration offers high efficiency, selectivity, and the ability to handle large volumes, making it a versatile tool for a wide range of applications.
Optimizing ultrafiltration workflows can enhance reproducibility, accuracy, and research productivity.
It involves passing the solution through a semipermeable membrane, allowing smaller molecules and solvents to pass through while retaining larger molecules and particles.
This process is commonly used in biomedical research, pharmaceutical development, and water treatment to isolate and concentrate biomolecules, proteins, and other macromolecules.
Ultrafiltration offers high efficiency, selectivity, and the ability to handle large volumes, making it a versatile tool for a wide range of applications.
Optimizing ultrafiltration workflows can enhance reproducibility, accuracy, and research productivity.
Most cited protocols related to «Ultrafiltration»
Ampicillin
Buffers
Chromatography, Affinity
Cloning Vectors
Culture Media
Deoxyribonuclease EcoRI
Escherichia coli
Freezing
imidazole
Oligonucleotide Primers
Open Reading Frames
Proteins
sodium phosphate
Strains
Tromethamine
Ultrafiltration
All fluorescent protein coding sequences were inserted between BamHI and EcoRI sites in the constitutive expression vector pNCS which encodes an N-terminal 6×His tag and linker. Sequences for all primers used in this study are listed in Supplementary Table 4 . Fluorescent proteins were expressed in E. coli strain NEBTurbo (New England Biolabs) or Mach1 (Invitrogen) by growing cultures in 2×YT medium supplemented with ampicillin overnight at 37 °C and shaking at 250 rpm. Fluorescent proteins were purified by Ni2+-affinity chromatography as previously described9 (link). Proteins were eluted in 50 mM Tris pH 7.5 or 50 mM sodium phosphate buffer pH 7.5 containing 250 mM imidazole. For all further characterization experiments, eluted fluorescent proteins were buffer-exchanged using Amicon Ultra0.5 10 kD MWCO ultrafiltration units (Millipore) into the same buffer without imidazole. Proteins were found to be stable when stored at 4°C indefinitely or when frozen at −20 °C or −80 °C.
Ampicillin
Buffers
Chromatography, Affinity
Cloning Vectors
Culture Media
Deoxyribonuclease EcoRI
Escherichia coli
Freezing
imidazole
Oligonucleotide Primers
Open Reading Frames
Proteins
sodium phosphate
Strains
Tromethamine
Ultrafiltration
The American Type Culture Collection (ATCC) provided the genomic DNA used to clone Acel_2062 (ATCC Number: ATCC 43068). Protein production and crystallization of the Acel_2062 protein was carried out by standard JCSG protocols
[8 (link)]. Clones were generated using the Polymerase Incomplete Primer Extension (PIPE) cloning method
[9 (link)]. The gene encoding Acel_2062 (GenBank: YP_873820[GenBank:YP_873820]; UniProtKB: A0LWM4[UniProtKB:A0LWM4]) was synthesized with codons optimized for Escherichia coli expression (Codon Devices, Cambridge, MA) and cloned into plasmid pSpeedET, which encodes an expression and purification tag followed by a tobacco etch virus (TEV) protease cleavage site (MGSDKIHHHHHHENLYFQ/G) at the amino terminus of the full-length protein. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed and dispensed on selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. Expression was performed in a selenomethionine-containing medium at 37°C. Selenomethionine was incorporated via inhibition of methionine biosynthesis
[10 (link)], which does not require a methionine auxotrophic strain. At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 μg/ml, and the cells were harvested and frozen. After one freeze/thaw cycle the cells were homogenized in lysis buffer [50 mM HEPES, 50 mM NaCl, 10 mM imidazole, 1 mM Tris(2-carboxyethyl)phosphine-HCl (TCEP), pH 8.0] and passed through a Microfluidizer (Microfluidics). The lysate was clarified by centrifugation at 32,500 x g for 30 minutes and loaded onto a nickel-chelating resin (GE Healthcare) pre-equilibrated with lysis buffer, the resin was washed with wash buffer [50 mM HEPES, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0], and the protein was eluted with elution buffer [20 mM HEPES, 300 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0]. The eluate was buffer exchanged with TEV buffer [20 mM HEPES, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP, pH 8.0] using a PD-10 column (GE Healthcare), and incubated with 1 mg of TEV protease per 15 mg of eluted protein for 2 hours at 20°–25°C followed by overnight at 4°C. The protease-treated eluate was passed over nickel-chelating resin (GE Healthcare) pre-equilibrated with HEPES crystallization buffer [20 mM HEPES, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP, pH 8.0] and the resin was washed with the same buffer. The flow-through and wash fractions were combined and concentrated to 15.6 mg/ml by centrifugal ultrafiltration (Millipore) for crystallization trials.
[8 (link)]. Clones were generated using the Polymerase Incomplete Primer Extension (PIPE) cloning method
[9 (link)]. The gene encoding Acel_2062 (GenBank: YP_873820[GenBank:YP_873820]; UniProtKB: A0LWM4[UniProtKB:A0LWM4]) was synthesized with codons optimized for Escherichia coli expression (Codon Devices, Cambridge, MA) and cloned into plasmid pSpeedET, which encodes an expression and purification tag followed by a tobacco etch virus (TEV) protease cleavage site (MGSDKIHHHHHHENLYFQ/G) at the amino terminus of the full-length protein. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed and dispensed on selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. Expression was performed in a selenomethionine-containing medium at 37°C. Selenomethionine was incorporated via inhibition of methionine biosynthesis
[10 (link)], which does not require a methionine auxotrophic strain. At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 μg/ml, and the cells were harvested and frozen. After one freeze/thaw cycle the cells were homogenized in lysis buffer [50 mM HEPES, 50 mM NaCl, 10 mM imidazole, 1 mM Tris(2-carboxyethyl)phosphine-HCl (TCEP), pH 8.0] and passed through a Microfluidizer (Microfluidics). The lysate was clarified by centrifugation at 32,500 x g for 30 minutes and loaded onto a nickel-chelating resin (GE Healthcare) pre-equilibrated with lysis buffer, the resin was washed with wash buffer [50 mM HEPES, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0], and the protein was eluted with elution buffer [20 mM HEPES, 300 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0]. The eluate was buffer exchanged with TEV buffer [20 mM HEPES, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP, pH 8.0] using a PD-10 column (GE Healthcare), and incubated with 1 mg of TEV protease per 15 mg of eluted protein for 2 hours at 20°–25°C followed by overnight at 4°C. The protease-treated eluate was passed over nickel-chelating resin (GE Healthcare) pre-equilibrated with HEPES crystallization buffer [20 mM HEPES, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP, pH 8.0] and the resin was washed with the same buffer. The flow-through and wash fractions were combined and concentrated to 15.6 mg/ml by centrifugal ultrafiltration (Millipore) for crystallization trials.
Agar
Anabolism
Buffers
Cells
Centrifugation
Codon
Crystallization
Cytokinesis
Escherichia coli
Fermentation
Freezing
G-substrate
Genes
Genome
Glycerin
GTP-Binding Proteins
HEPES
imidazole
Medical Devices
Methionine
Muramidase
Nickel
Oligonucleotide Primers
Peptide Hydrolases
phosphine
Plasmids
Proteins
Psychological Inhibition
Resins, Plant
Selenomethionine
Sodium Chloride
Strains
TEV protease
Tobacco etch virus
tris(2-carboxyethyl)phosphine
Tromethamine
Ultrafiltration
2-Mercaptoethanol
Acetic Acid
Biological Assay
Cells
Centrifugation
Chloroform
Chromatography
Deoxycholic Acid, Monosodium Salt
Digestion
Disgust
Edetic Acid
Electrostatics
Ethylmaleimide
formic acid
Glycerin
Guanidine
Hydrophilic Interactions
Liquid Chromatography
M-200
Medical Devices
Methanol
Neoplasm Metastasis
Peptides
Proteins
Radionuclide Imaging
Sodium Chloride
Sulfhydryl Compounds
Tandem Mass Spectrometry
tris(2-carboxyethyl)phosphine
Tromethamine
Ultrafiltration
A 480-nt DNA fragment comprising the rnhA open reading frame (ORF) was amplified from M. smegmatis genomic DNA by PCR with primers that introduced a NdeI site at the start codon and a BglII site immediately after the stop codon. The PCR product was digested with NdeI and BglII and inserted between the NdeI and BamHI sites of pET16b-His10 to generate expression plasmid pET-His10•RnhA. Missense mutations E50Q and D72N were introduced into the rnhC ORF by PCR with mutagenic primers. The inserts in each plasmid were sequenced to verify the fusion junctions and to ensure that no unwanted coding changes were introduced during amplification and cloning.
The RnhA expression plasmids were transfected into E. coli BL21 (DE3) cells. Cultures (2-liter) amplified from single transformants were grown at 37°C in Terrific Broth containing 100 μg/ml ampicillin until the A600 reached 0.8. The cultures were chilled on ice for 1 h, adjusted to 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG), and then incubated for 20 h at 18°C with constant shaking. All subsequent steps of purification were performed at 4°C. Cells were harvested by centrifugation and resuspended in 35 ml of buffer A (50 mM Tris–HCl, pH 7.5, 500 mM NaCl, 20 mM imidazole, 10% glycerol). The cells were lysed by sonication and the insoluble material was removed by centrifugation at 18000 rpm for 45 min. Supernatants were mixed for 1 h with 4 ml of Ni-NTA resin (Qiagen) that had been equilibrated with buffer A. The resins were poured into gravity-flow columns and then washed with 60 ml of buffer A. The adsorbed proteins were step-eluted with 300 mM imidazole in buffer A. The polypeptide compositions of the eluate fractions were monitored by SDS-PAGE and the peak fractions containing each recombinant protein were pooled. RnhA proteins were then subjected to gel filtration through a Superdex-200 column (GE Healthcare) equilibrated in 50 mM Tris–HCl, pH 7.5, 200 mM NaCl, 10% glycerol. Peak monomeric RnhA fractions were pooled, concentrated by centrifugal ultrafiltration to 23 mg/ml [wild-type RnhA], 12 mg/ml [RnhC-E50Q], and 13 mg/ml [RnhC-D72N] and then stored at −80°C. Protein concentrations were determined from the A280 measured with a Nanodrop spectrophotometer (Thermo scientific), applying extinction coefficients calculated with Protparam.
The RnhA expression plasmids were transfected into E. coli BL21 (DE3) cells. Cultures (2-liter) amplified from single transformants were grown at 37°C in Terrific Broth containing 100 μg/ml ampicillin until the A600 reached 0.8. The cultures were chilled on ice for 1 h, adjusted to 0.5 mM isopropyl-β-
Ampicillin
Buffers
Cells
Centrifugation
Codon, Initiator
Codon, Terminator
Escherichia coli
Extinction, Psychological
Gel Chromatography
Genome
Glycerin
Gravity
imidazole
Missense Mutation
Mutagenesis
Oligonucleotide Primers
Plasmids
Polypeptides
Proteins
Recombinant Proteins
Resins, Plant
SDS-PAGE
Sodium Chloride
Tromethamine
Ultrafiltration
Most recents protocols related to «Ultrafiltration»
Example 1
The sequence coding for the light chain variable region of the antibody was inserted into vector pFUSE2ss-CLIg-hK (Invivogen, Catalog Number: pfuse2ss-hclk) using EcoRI and BsiWI restriction sites to construct a light chain expression vector. The sequence coding for the heavy chain variable region of the antibody was inserted into vector pFUSEss-CHIg-hG2 (Invivogen, Catalog Number: pfusess-hchg2) or vector pFUSEss-CHIg-hG4 (Invivogen, Catalog Number: pfusess-hchg4) using EcoRI and NheI restriction sites to construct a heavy chain expression vector.
The culture and transfection of Expi293 cells were performed in accordance with the handbook of Expi293™ Expression System Kit from Invitrogen (Catalog Number: A14635). The density of the cells was adjusted to 2×106 cells/ml for transfection, and 0.6 μg of the light chain expression vector as described above and 0.4 μg of the heavy chain expression vector as described above were added to each ml of cell culture, and the supernatant of the culture was collected four days later.
The culture supernatant was subjected to non-reduced SDS-PAGE gel electrophoresis in accordance with the protocol described in Appendix 8, the Third edition of the “Molecular Cloning: A Laboratory Manual”.
Pictures were taken with a gel scanning imaging system from BEIJING JUNYI Electrophoresis Co., LTD and in-gel quantification was performed using Gel-PRO ANALYZER software to determine the expression levels of the antibodies after transient transfection. Results were expressed relative to the expression level of control antibody 1 (control antibody 1 was constructed according to U.S. Pat. No. 7,186,809, which comprises a light chain variable region as set forth in SEQ ID NO: 10 of U.S. Pat. No. 7,186,809 and a heavy chain variable region as set forth in SEQ ID NO: 12 of U.S. Pat. No. 7,186,809, the same below) (control antibody 2 was constructed according to U.S. Pat. No. 7,638,606, which comprises a light chain variable region as set forth in SEQ ID NO: 6 of U.S. Pat. No. 7,638,606 and a variable region as set forth in SEQ ID NO: 42 of U.S. Pat. No. 7,638,606, the same below). See Tables 2a-2c below for the results.
Example 4
6-8 week-old SPF Balb/c mice were selected and injected subcutaneously with antibodies (the antibodies of the present invention or control antibody 2) in a dose of 5 mg/kg (weight of the mouse). Blood samples were collected at the time points before administration (0 h) and at 2, 8, 24, 48, 72, 120, 168, 216, 264, 336 h after administration. For blood sampling, the animals were anesthetized by inhaling isoflurane, blood samples were taken from the orbital venous plexus, and the sampling volume for each animal was about 0.1 ml; 336 h after administration, the animals were anesthetized by inhaling isoflurane and then euthanized after taking blood in the inferior vena cava.
No anticoagulant was added to the blood samples, and serum was isolated from each sample by centrifugation at 1500 g for 10 min at room temperature within 2 h after blood sampling. The collected supernatants were immediately transferred to new labeled centrifuge tubes and then stored at −70° C. for temporary storage. The concentrations of the antibodies in the mice were determined by ELISA:
1. Preparation of Reagents
sIL-4Rα (PEPRO TECH, Catalog Number: 200-04R) solution: sIL-4Rα was taken and 1 ml ddH2O was added therein, mixed up and down, and then a solution of 100 μg/ml was obtained. The solution was stored in a refrigerator at −20° C. after being subpacked.
Sample to be tested: 1 μl of serum collected at different time points was added to 999 μl of PBS containing 1% BSA to prepare a serum sample to be tested of 1:1000 dilution.
Standard sample: The antibody to be tested was diluted to 0.1 μg/ml with PBS containing 1% BSA and 0.1% normal animal serum (Beyotime, Catalog Number: ST023). Afterwards, 200, 400, 600, 800, 900, 950, 990 and 1000 μl of PBS containing 1% BSA and 0.1% normal animal serum were respectively added to 800, 600, 400, 200, 100, 50, 10 and 0 μl of 0.1 μg/ml antibodies to be tested, and thus standard samples of the antibodies of the present invention were prepared with a final concentration of 80, 60, 40, 20, 10, 5, 1, or 0 ng/ml respectively.
2. Detection by ELISA
250 μl of 100 μg/ml sIL-4Rα solution was added to 9.75 ml of PBS, mixed up and down, and then an antigen coating buffer of 2.5 μg/ml was obtained. The prepared antigen coating buffer was added to a 96-well ELISA plate (Corning) with a volume of 100 μl per well. The 96-well ELISA plate was incubated overnight in a refrigerator at 4° C. after being wrapped with preservative film (or covered). On the next day, the 96-well ELISA plate was taken out and the solution therein was discarded, and PBS containing 2% BSA was added thereto with a volume of 300 μl per well. The 96-well ELISA plate was incubated for 2 hours in a refrigerator at 4° C. after being wrapped with preservative film (or covered). Then the 96-well ELISA plate was taken out and the solution therein was discarded, and the plate was washed 3 times with PBST. The diluted standard antibodies and the sera to be detected were sequentially added to the corresponding wells, and three duplicate wells were made for each sample with a volume of 100 μl per well. The ELISA plate was wrapped with preservative film (or covered) and incubated for 1 h at room temperature. Subsequently, the solution in the 96-well ELISA plate was discarded and then the plate was washed with PBST for 3 times. Later, TMB solution (Solarbio, Catalog Number: PR1200) was added to the 96-well ELISA plate row by row with a volume of 100 μl per well. The 96-well ELISA plate was placed at room temperature for 5 minutes, and 2 M H2SO4 solution was added in immediately to terminate the reaction. The 96-well ELISA plate was then placed in flexstation 3 (Molecular Devices), the values of OD450 were read, the data were collected and the results were calculated with Winnonlin software. The pharmacokinetic results were shown in
Example 5
A series of pharmacokinetic experiments were carried out in Macaca fascicularises to further screen antibodies.
3-5 year-old Macaca fascicularises each weighting 2-5 Kg were selected and injected subcutaneously with antibodies (the antibodies of the present invention or control antibody 2) in a dose of 5 mg/kg (weight of the Macaca fascicularis). The antibody or control antibody 2 to be administered was accurately extracted with a disposable aseptic injector, and multi-point injections were made subcutaneously on the inner side of the thigh of the animal, and the injection volume per point was not more than 2 ml. Whole blood samples were collected from the subcutaneous vein of the hind limb of the animal at the time points before administration (0 h) and at 0.5, 2, 4, 8, 24, 48, 72, 120, 168, 240, 336 h, 432 h, 504 h, 600 h, 672 h after administration. The blood volume collected from each animal was about 0.1 ml each time.
No anticoagulant was added to the blood samples, and serum was isolated from each sample by centrifugation at 1500 g for 10 min at room temperature within 2 h after blood sampling. The collected supernatants were immediately transferred to new labeled centrifuge tubes and then stored at −70° C. for temporary storage. The concentrations of the antibodies in the Macaca fascicularises were determined according the method as described in Example 4. The pharmacokinetic results are shown in
Example 10
In vivo pharmacokinetics of the antibodies of the invention are further detected and compared in this Example, in order to investigate the possible effects of specific amino acids at specific positions on the pharmacokinetics of the antibodies in animals. The specific experimental method was the same as that described in Example 4, and the results are shown in Table 9 below.
From the specific sequence, the amino acid at position 103 in the sequence of the heavy chain H1031 (SEQ ID NO. 91) of the antibody (in CDR3) is Asp (103Asp), and the amino acid at position 104 is Tyr (104Tyr). Compared with antibodies that have no 103Asp and 104Tyr in heavy chain, the present antibodies which have 103Asp and 104Tyr have a 2- to 4-fold higher area under the drug-time curve and an about 70% reduced clearance rate.
The expression levels of the antibodies of the present invention are also detected and compared, in order to investigate the possible effects of specific amino acids at specific positions on the expression of the antibodies. Culture and transfection of Expi293 cells were conducted according to Example 1, and the collected culture supernatant was then passed through a 0.22 μm filter and then purified by GE MabSelect Sure (Catalog Number: 11003494) Protein A affinity chromatography column in the purification system GE AKTA purifier 10. The purified antibody was collected and concentrated using Amicon ultrafiltration concentrating tube (Catalog Number: UFC903096) and then quantified. The quantitative results are shown in Table 10 below.
From the specific sequence, the amino acid at position 31 in the sequence of the light chain L1012 (SEQ ID NO. 44), L1020 (SEQ ID NO. 55) or L1023 (SEQ ID NO. 51) of the antibody (in CDR1) is Ser (31Ser). Compared with antibodies that have no 31Ser in light chain, the present antibodies which have 31Ser have a 2- to 5-fold higher expression level.
The above description for the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes and variations according to the present invention, which are within the protection scope of the claims of the present invention without departing from the spirit of the same.
Amino Acids
Animals
Antibodies
Anticoagulants
Antigens
Asepsis
BLOOD
Blood Volume
Buffers
Cell Culture Techniques
Cells
Centrifugation
Chromatography
Chromatography, Affinity
Cloning Vectors
Culture Media
Deoxyribonuclease EcoRI
Drug Kinetics
Electrophoresis
Enzyme-Linked Immunosorbent Assay
Hindlimb
Human Body
Immunoglobulin Heavy Chains
Immunoglobulin Light Chains
Immunoglobulins
Interleukin-1
Isoflurane
Light
Macaca
Macaca fascicularis
Medical Devices
Metabolic Clearance Rate
Mice, Inbred BALB C
Mus
Open Reading Frames
Pharmaceutical Preparations
Pharmaceutical Preservatives
SDS-PAGE
Serum
Staphylococcal Protein A
Technique, Dilution
Thigh
Transfection
Transients
Ultrafiltration
Veins
Vena Cavas, Inferior
Example 1
An exemplary process for producing MSC-derived exosomes is shown in
Cells
Chromatography
Exosomes
Homo sapiens
Safety
Strains
Tissues
Ultrafiltration
Example 5
Using N-succinimidyl-3-(4-hydroxy-3-[125I]iodophenyl)propionate, Bolton-Hunter Reagent (NEX120, PerkinElmer), the purified ST03-Cupid protein was radio-iodinated. Specifically, Bolton-Hunter Reagent in an amount of 20 times the number of moles of the used protein was taken in an Eppendorf tube, the solvent was then vaporized, and the protein solution was then added thereto. The mixture was reacted on ice for 2 hours. Thereafter, the reaction mixture was subjected to a desalination column (PD MiniTrap G-25, GE Healthcare) to remove unreacted radio-iodination Bolton-Hunter Reagent, and the resultant was then subjected to an ultrafiltration column (VIVASPIN Turbo 4, Sartorius) for concentration. After completion of the concentration, the amount of radioactivity was measured using a dose calibrator (CRC-25w, CAPINTEC), and the quality of the protein was checked by performing CBB staining according to SDS-PAGE.
Bolton-Hunter reagent
Iodination
Nevus
Propionate
Proteins
Radioactivity
SDS-PAGE
Solvents
Ultrafiltration
Example 1
Fermentation/Concentration
In some embodiments, whey permeate, concentrated permeate, and/or ultrafiltration permeate is pasteurized and then fermented with Lactic acid bacteria for 20 to 30 hours at 10-130° F. with injection of NH4(OH) to maintain pH at 5.5 to 5.6 during fermentation. The resulting fermented liquid is concentrated by mechanical vapor recompression (MVR) to achieve a solids content of about 58%-64%. The concentrated fermented liquid is then sent to a pH balance tank where it is injected with NH4(OH) to achieve a pH of about 6.5 to 6.7.
Crystallization
The concentrated fermented liquid is then sent to a plate heat exchanger (PHE) to bring the temperature of the liquid to about 130° F. The concentrated fermented liquid is then sent to a crystallization tank where the concentrated fermented liquid is agitated and allowed to cool to about 110° F. to 115° F., during which crystal formation occurs. In some embodiments, once the temperature of the concentrated fermented liquid reaches about 90° F. to 115° F. the concentrated fermented liquid is sent to a decanter centrifuge to separate the solid crystals from the liquid. Across 12 fermentation batches from production, the average yield of solid crystals was 1,744 lb.
Across multiple processing trials the following crystal yields were achieved:
Example 2
Fermentation/Concentration
In some embodiments, whey permeate, concentrated permeate, and/or ultrafiltration permeate is pasteurized and then fermented with Lactic acid bacteria for 20 to 30 hours at 100-120° F. with injection of NH4(OH) to maintain pH at 5.5 to 5.6. The resulting fermented liquid is concentrated by mechanical vapor recompression (MVR) to achieve a solids content of about 61%-64%.
Crystallization
The concentrated fermented liquid is then sent directly to a crystallizer tank with continuous agitation. In this example, the liquid is not sent to pH balance tank or chiller plate heat exchanger. To achieve higher crystal yield, a 3000 (w/w) CaOH slurry is added to the concentrated fermented liquid in the crystallization tank to achieve a calcium concentration of 0.9-2.0% (w/w) in the combined mixture. The CaOH slurry is added to the concentrated fermented liquid in the crystallizer tank slowly to allow thorough mixing. The mixture is then allowed to stand in the crystallization tank for 6 to 18 hours, during which time the temperature is allowed to cool to about 90 to 115° F. and crystals are formed. Once the temperature of the concentrated fermented liquid reaches about 90 to 115° F. the concentrated fermented liquid is sent to a decanter to separate the solid crystals from the liquid.
Across multiple processing trials the following crystal yields were achieved with a calcium concentration of 3.33% (non-seeded data from Example 1 is included for comparison):
Calcium, Dietary
Crystallization
Fermentation
Hydroxide, Calcium
Lactobacillales
Liquid Crystals
TO 115
Ultrafiltration
Whey
Example 5
A variety of % silk concentrations have been produced through the use of Tangential Flow Filtration (TFF). In all cases a 1% silk solution was used as the input feed. A range of 750-18,000 mL of 1% silk solution was used as the starting volume. Solution is diafiltered in the TFF to remove lithium bromide. Once below a specified level of residual LiBr, solution undergoes ultrafiltration to increase the concentration through removal of water. See examples below.
7.30% Silk Solution: A 7.30% silk solution was produced beginning with 30 minute extraction batches of 100 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 100 g of silk fibers were dissolved per batch to create 20% silk in LiBr. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 15,500 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 1300 mL. 1262 mL of 7.30% silk was then collected. Water was added to the feed to help remove the remaining solution and 547 mL of 3.91% silk was then collected.
6.44% Silk Solution: A 6.44% silk solution was produced beginning with 60 minute extraction batches of a mix of 25, 33, 50, 75 and 100 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 35, 42, 50 and 71 g per batch of silk fibers were dissolved to create 20% silk in LiBr and combined. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 17,000 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 3000 mL. 1490 mL of 6.44% silk was then collected. Water was added to the feed to help remove the remaining solution and 1454 mL of 4.88% silk was then collected
2.70% Silk Solution: A 2.70% silk solution was produced beginning with 60 minute extraction batches of 25 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100° C. 9.3 M LiBr in a 100° C. oven for 1 hour. 35.48 g of silk fibers were dissolved per batch to create 20% silk in LiBr. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 um filter to remove large debris. 1000 mL of 1%, filtered silk solution was used as the starting volume/diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 300 mL. 312 mL of 2.7% silk was then collected.
Filtration
lithium bromide
Silk
Solvents
Ultrafiltration
Top products related to «Ultrafiltration»
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The Amicon Ultra is a centrifugal filter device used for the concentration and purification of macromolecules such as proteins, peptides, and nucleic acids. It operates by applying centrifugal force to separate the sample components based on their molecular weight.
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The Amicon Ultra-15 Centrifugal Filter Units are a type of laboratory equipment used for the concentration and purification of macromolecules, such as proteins, DNA, and other biological samples. These units employ a centrifugal force to pass the sample through a semi-permeable membrane, allowing the desired molecules to be retained while smaller components are filtered out.
Sourced in Germany, United Kingdom, Hungary, Japan
The Vivaspin 500 is a centrifugal concentrator device used for the concentration and desalting of biological samples. It features a spin column design with a molecular weight cut-off membrane that retains the sample while allowing smaller molecules to pass through during centrifugation.
Sourced in United States, Germany, Ireland, Italy, United Kingdom
The Amicon Ultra-4 is a centrifugal filter device designed for the concentration and desalting of protein and other macromolecular solutions. It is capable of processing sample volumes up to 4 milliliters.
Sourced in United States, Germany
The Ultrafiltration centrifuge tube is a laboratory equipment designed for the separation and concentration of macromolecules, such as proteins, nucleic acids, and other biological samples. It utilizes a semi-permeable membrane to selectively filter and retain the desired molecules while allowing smaller components to pass through during the centrifugation process.
More about "Ultrafiltration"
Ultrafiltration is a powerful separation technique used to purify, concentrate, and isolate biomolecules, proteins, and other macromolecules.
This process involves passing a solution through a semipermeable membrane, allowing smaller molecules and solvents to pass through while retaining larger molecules and particles.
This high-efficiency, selective method is commonly employed in biomedical research, pharmaceutical development, and water treatment applications.
Amicon Ultra and Amicon Ultra-15 are leading ultrafiltration devices that utilize centrifugal force to concentrate samples.
These Amicon Ultra-15 Centrifugal Filter Units are versatile tools that can handle a wide range of sample volumes.
Similarly, Vivaspin 500 and Amicon Ultra-4 are other ultrafiltration tube options that enable efficient sample preparation and processing.
Ultrafiltration workflows are often integrated with enzymatic digestion, such as Trypsin, to facilitate the purification and analysis of proteins and peptides.
By optimizing these ultrafiltration procedures, researchers can enhance reproducibility, accuracy, and overall research productivity.
PubCompare.ai is an AI-driven platform that can help locate the best ultrafiltration protocols from literature, preprints, and patents, allowing you to identify the optimal products and procedures to elevate your research.
Experience the power of PubCompare.ai and take your ultrafiltration studies to new heights.
This process involves passing a solution through a semipermeable membrane, allowing smaller molecules and solvents to pass through while retaining larger molecules and particles.
This high-efficiency, selective method is commonly employed in biomedical research, pharmaceutical development, and water treatment applications.
Amicon Ultra and Amicon Ultra-15 are leading ultrafiltration devices that utilize centrifugal force to concentrate samples.
These Amicon Ultra-15 Centrifugal Filter Units are versatile tools that can handle a wide range of sample volumes.
Similarly, Vivaspin 500 and Amicon Ultra-4 are other ultrafiltration tube options that enable efficient sample preparation and processing.
Ultrafiltration workflows are often integrated with enzymatic digestion, such as Trypsin, to facilitate the purification and analysis of proteins and peptides.
By optimizing these ultrafiltration procedures, researchers can enhance reproducibility, accuracy, and overall research productivity.
PubCompare.ai is an AI-driven platform that can help locate the best ultrafiltration protocols from literature, preprints, and patents, allowing you to identify the optimal products and procedures to elevate your research.
Experience the power of PubCompare.ai and take your ultrafiltration studies to new heights.