> Procedures > Laboratory Procedure > Centrifugation

Centrifugation

Q: What are the different types or variations of centrifugation techniques?

Centrifugation techniques can vary in terms of speed, duration, temperature, and rotor type, depending on the specific application. Common types include differential centrifugation, density gradient centrifugation, and continuous-flow centrifugation, each with their own unique applications and considerations.

Centrifugation is a widely used technique in biomedical research and clinical diagnostics.
It involves the separation of particles, cells, or molecules based on their differential sedimentation rates within a centrifugal force field.
This process allows for the purification, concentration, or isolation of various biological components, such as proteins, nucleic acids, organelles, and cells.
Centrifugation protocols can vary in terms of speed, duration, temperature, and rotor type, depending on the specific application and sample characteristics.
Optimizing these parameters is crucial for ensuring the reproducibility and reliability of experimental results.
PubCompare.ai offers an AI-powered platform to help researchers easily locate, compare, and identify the best centrifugation methods and products to enhance their research workflows and improve data quality.
Leveraging the power of artificial intelligence, PubCompare.ai can help take your work to the next level by providing data-driven insights and recommendations to optimize your centrifugation protocols.

Most cited protocols related to «Centrifugation»

Protein-Protein Interaction Assay

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.

Publication 2017

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

Quantitative Proteomics of HeLa-E. coli Mixture

An Escherichia coli K12 strain was grown in standard LB medium, harvested, washed in PBS, and lysed in BugBuster (Novagen Merck Chemicals, Schwalbach, Germany) according to the manufacturer's protocol. HeLa S3 cells were grown in standard RPMI 1640 medium supplemented with glutamine, antibiotics, and 10% FBS. After being washed with PBS, cells were lysed in cold modified RIPA buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 150 mm NaCl, 1% N-octylglycoside, 0.1% sodium deoxycholate, complete protease inhibitor mixture (Roche)) and incubated for 15 min on ice. Lysates were cleared by centrifugation, and after precipitation with chloroform/methanol, proteins were resuspended in 6 m urea, 2 m thiourea, 10 mm HEPES, pH 8.0. Prior to in-solution digestion, 60-μg protein samples from HeLa S3 lysates were spiked with either 10 μg or 30 μg of E. coli K12 lysates based on protein amount (Bradford assay). Both batches were reduced with dithiothreitol and alkylated with iodoacetamide. Proteins were digested with LysC (Wako Chemicals, GmbH, Neuss, Germany) for 4 h and then trypsin digested overnight (Promega, GmbH, Mannheim, Germany). Digestion was stopped by the addition of 2% trifluroacetic acid. Peptides were separated by isoelectric focusing into 24 fractions on a 3100 OFFGEL Fractionator (Agilent, GmbH, Böblingen, Germany) as described in Ref. 45 (link). Each fraction was purified with C₁₈ StageTips (46 (link)) and analyzed via liquid chromatography combined with electrospray tandem mass spectrometry on an LTQ Orbitrap (Thermo Fisher) with lock mass calibration (47 (link)). All raw files were searched against the human and E. coli complete proteome sequences obtained from UniProt (version from January 2013) and a set of commonly observed contaminants. MS/MS spectra were filtered to contain at most eight peaks per 100 mass unit intervals. The initial MS mass tolerance was 20 ppm, and MS/MS fragment ions could deviate by up to 0.5 Da (48 (link)). For quantification, intensities can be determined alternatively as the full peak volume or as the intensity maximum over the retention time profile, and the latter method was used here as the default. Intensities of different isotopic peaks in an isotope pattern are always summed up for further analysis. MaxQuant offers a choice of the degree of uniqueness required in order for peptides to be included for quantification: “all peptides,” “only unique peptides,” and “unique plus razor peptides” (42 (link)). Here we chose the latter, because it is a good compromise between the two competing interests of using only peptides that undoubtedly belong to a protein and using as many peptide signals as possible. The distribution of peptide ions over fractions and samples is shown in supplemental Fig. S1.

Cox J., Hein M.Y., Luber C.A., Paron I., Nagaraj N, & Mann M. (2014). Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ. Molecular & Cellular Proteomics : MCP, 13(9), 2513-2526.

Publication 2014

Acids Antibiotics, Antitubercular Biological Assay Buffers Cells Centrifugation Chloroform Cold Temperature Deoxycholic Acid, Monosodium Salt Digestion Dithiothreitol Edetic Acid Escherichia coli Escherichia coli K12 Glutamine HeLa Cells HEPES Homo sapiens Immune Tolerance Iodoacetamide Ions Isotopes Liquid Chromatography Methanol Peptides Promega Protease Inhibitors Proteins Proteome Radioimmunoprecipitation Assay Retention (Psychology) Sodium Chloride Staphylococcal Protein A Tandem Mass Spectrometry Thiourea Tromethamine Trypsin Urea

Nuclei Isolation for ATAC-Seq Analysis

See Supplementary
Protocol 2 for a detailed protocol. This protocol is highly similar
to the INTACT method^{19 (link)} and
either protocol can be used for the isolation of nuclei with equivalent results.
All of the steps were carried out at 4 °C. A frozen tissue fragment ~20
mg was placed into a pre-chilled 2-ml Dounce homogenizer containing 2 ml of cold
1× homogenization buffer (320 mM sucrose, 0.1 mM EDTA, 0.1%
NP40, 5 mM CaCl₂, 3 mM Mg(Ac)₂, 10 mM Tris pH 7.8,
1× protease inhibitors (Roche, cOmplete), and 167 μM
β-mercaptoethanol, in water). Tissue was homogenized with approximately
ten strokes with the loose ‘A’ pestle, followed by 20 strokes
with the tight ‘B’ pestle. Connective tissue and residual debris
were precleared by filtration through an 80-μm nylon mesh filter
followed by centrifugation for 1 min at 100 r.c.f. While avoiding the pelleted
debris, 400 μl was transferred to a pre-chilled 2-ml round bottom
Lo-Bind Eppendorf tube. An equal volume (400 μl) of a 50%
iodixanol solution (50% iodixanol in 1× homogenization buffer)
was added and mixed by pipetting to make a final concentration of 25%
iodixanol. 600 μl of a 29% iodixanol solution (29%
iodixanol in 1× homogenization buffer containing 480 mM sucrose) was
layered underneath the 25% iodixanol mixture. A clearly defined
interface should be visible. In a similar fashion, 600 μl of a
35% iodixanol solution (35% iodixanol in 1×
homogenization containing 480 mM sucrose) was layered underneath the 29%
iodixanol solution. Again, a clearly defined interface should be visible between
all three layers. In a swinging-bucket centrifuge, nuclei were centrifuged for
20 min at 3,000 r.c.f. After centrifugation, the nuclei were present at the
interface of the 29% and 35% iodixanol solutions. This band with
the nuclei was collected in a 300 μl volume and transferred to a
pre-chilled tube. Nuclei were counted after addition of trypan blue, which
stains all nuclei due to membrane permeabilization from freezing. 50,000 counted
nuclei were then transferred to a tube containing 1 ml of ATAC-seq RSB with
0.1% Tween-20. Nuclei were pelleted by centrifugation at 500 r.c.f. for
10 min in a pre-chilled (4 °C) fixed-angle centrifuge. Supernatant was
removed using the two pipetting steps described above. Because the nuclei were
already permeabilized, no lysis step was performed, and the transposition mix
(25 μl 2× TD buffer, 2.5 μl transposase (100 nM final),
16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl
10% Tween-20, 5 μl water) was added directly to the nuclear
pellet and mixed by pipetting up and down six times. Transposition reactions
were incubated at 37 °C for 30 min in a thermomixer with shaking at
1,000 r.p.m. Reactions were cleaned up with Zymo DNA Clean and Concentrator 5
columns. The remainder of the ATAC-seq library preparation was performed as
described previously¹⁸.

Corces M.R., Trevino A.E., Hamilton E.G., Greenside P.G., Sinnott-Armstrong N.A., Vesuna S., Satpathy A.T., Rubin A.J., Montine K.S., Wu B., Kathiria A., Cho S.W., Mumbach M.R., Carter A.C., Kasowski M., Orloff L.A., Risca V.I., Kundaje A., Khavari P.A., Montine T.J., Greenleaf W.J, & Chang H.Y. (2017). An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nature methods, 14(10), 959-962.

Publication 2017

2-Mercaptoethanol ATAC-Seq Buffers Cell Nucleus Centrifugation Cerebrovascular Accident Connective Tissue Digitonin DNA Library Edetic Acid Filtration iodixanol isolation Nylons Protease Inhibitors Sucrose Tissue, Membrane Tissues Transposase Tromethamine Trypan Blue Tween 20

SARS-CoV-2 Spike Protein Expression Protocols

The mammalian cell codon-optimized nucleotide sequence coding for the spike protein of the SARS-CoV-2 isolate (GenBank:MN908947.3) was synthesized commercially (Genewiz). The RBD (amino acids 319–541; RVQP…CVNF), along with the signal peptide (amino acids 1–14; MFVF…VSSQ) plus a hexahistidine tag, was cloned into mammalian expression vector pCAGGS as well as in a modified pFastBac Dual vector for baculovirus system expression. The soluble version of the spike protein (amino acids 1–1,213; MFVF…IKWP), including a C-terminal thrombin cleavage site, T4 foldon trimerization domain and hexahistidine tag, was also cloned into pCAGGS. The protein sequence was modified to remove the polybasic cleavage site (RRAR to A), and two stabilizing mutations were introduced as well (K986P and V987P; wild-type numbering). Recombinant proteins were produced using the well-established baculovirus expression system and this system has been published in detail in refs.^{20 (link)–22}, including a video guide. Recombinant proteins were also produced in Expi293F cells (Thermo Fisher Scientific) by transfections of these cells with purified DNA using an ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Supernatants from transfected cells were harvested on day 3 post-transfection by centrifugation of the culture at 4,000g for 20 min. Supernatant was then incubated with 6 ml Ni-NTA Agarose (Qiagen) for 1–2 h at room temperature. Next, gravity flow columns were used to collect the Ni-NTA agarose and the protein was eluted. Each protein was concentrated in Amicon centrifugal units (EMD Millipore) and re-suspended in phosphate-buffered saline (PBS). Proteins were analyzed by reducing SDS-PAGE. The DNA sequence for all constructs is available from the Krammer Laboratory and has also been deposited in GenBank (additional information in the ‘Data availability’ statement). Several of the expression plasmids and proteins have also been submitted to the BEI Resources repository and can be requested from their web page for free (https://www.beiresources.org/. S1 proteins of NL63 and 229E were obtained from Sino Biological (produced in hexahistidine-tagged 293HEK cells). A detailed protocol for protein expression of RBD and spike in mammalian cells is also available^{7 (link)}.

Amanat F., Stadlbauer D., Strohmeier S., Nguyen T.H., Chromikova V., McMahon M., Jiang K., Arunkumar G.A., Jurczyszak D., Polanco J., Bermudez-Gonzalez M., Kleiner G., Aydillo T., Miorin L., Fierer D.S., Lugo L.A., Kojic E.M., Stoever J., Liu S.T., Cunningham-Rundles C., Felgner P.L., Moran T., García-Sastre A., Caplivski D., Cheng A.C., Kedzierska K., Vapalahti O., Hepojoki J.M., Simon V, & Krammer F. (2020). A serological assay to detect SARS-CoV-2 seroconversion in humans. Nature medicine, 26(7), 1033-1036.

Publication 2020

Amino Acids Amino Acid Sequence Baculoviridae Biopharmaceuticals Cells Centrifugation Cloning Vectors Codon Cytokinesis DNA Sequence Gravity His-His-His-His-His-His isononanoyl oxybenzene sulfonate Mammals M protein, multiple myeloma Mutation Open Reading Frames Phosphates Plasmids Proteins Recombinant Proteins Saline Solution SARS-CoV-2 SDS-PAGE Sepharose Signal Peptides Staphylococcal Protein A Thrombin Transfection

Isolation of Murine Cardiac Myocytes

A schematic overview of the myocyte isolation procedure is shown in Figure 2. An expanded description of the procedure, accompanied with images and videos, and complete materials list is available in the Online Data Supplement, alongside full details of additional methods applied in this study (Appendix A-ix). All animal work was undertaken in accordance with Singapore National Advisory Committee for Laboratory Animal Research guidelines. Relevant national and institutional guidelines and regulations must be consulted before commencement of all animal work.
Buffers and media were prepared as detailed in Appendix D. EDTA, perfusion, and collagenase buffers were apportioned into sterile 10 mL syringes, and sterile 27 G hypodermic needles were attached (Online Figure IA).
C57/BL6J mice aged 8 to 12 weeks were anesthetized, and the chest was opened to expose the heart. Descending aorta was cut, and the heart was immediately flushed by injection of 7 mL EDTA buffer into the right ventricle. Ascending aorta was clamped using Reynolds forceps, and the heart was transferred to a 60-mm dish containing fresh EDTA buffer. Digestion was achieved by sequential injection of 10 mL EDTA buffer, 3 mL perfusion buffer, and 30 to 50 mL collagenase buffer into the left ventricle (LV). Constituent chambers (atria, LV, and right ventricle) were then separated and gently pulled into 1-mm pieces using forceps. Cellular dissociation was completed by gentle trituration, and enzyme activity was inhibited by addition of 5 mL stop buffer.
Cell suspension was passed through a 100-μm filter, and cells underwent 4 sequential rounds of gravity settling, using 3 intermediate calcium reintroduction buffers to gradually restore calcium concentration to physiological levels. The cell pellet in each round was enriched with myocytes and ultimately formed a highly pure myocyte fraction, whereas the supernatant from each round was combined to produce a fraction containing nonmyocyte cardiac populations.
CM yields and percentage of viable rod-shaped cells were quantified using a hemocytometer. Where required, the CMs were resuspended in prewarmed plating media and plated at an applicationdependent density, onto laminin (5 μg/mL) precoated tissue culture plastic or glass coverslips, in a humidified tissue culture incubator (37°C, 5% CO₂). After 1 hour, and every 48 hours thereafter, media was changed to fresh, prewarmed culture media.
The cardiac nonmyocyte fraction was collected by centrifugation (300g, 5 minutes), resuspended in fibroblast growth media, and plated on tissue-culture treated plastic, area ≈ 23 cm² (0.5× 12-well plate) per LV, in a humidified tissue culture incubator. Media was changed after 24 hours and every 48 hours thereafter.

Ackers-Johnson M., Li P.Y., Holmes A.P., O’Brien S.M., Pavlovic D, & Foo R.S. (2016). A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart. Circulation research, 119(8), 909-920.

Publication 2016

Animals Animals, Laboratory Ascending Aorta Buffers Calcium Centrifugation Chest Collagenase Culture Media Descending Aorta Dietary Supplements Digestion Edetic Acid enzyme activity Fibroblasts Forceps Gravity Heart Heart Atrium Hyperostosis, Diffuse Idiopathic Skeletal Hypodermic Needles isolation Laminin Left Ventricles Mus Muscle Cells Perfusion physiology Population Group Retreatments Rod Photoreceptors Sterility, Reproductive Syringes Tissues Ventricles, Right

Most recents protocols related to «Centrifugation»

Rituximab-Loaded ABRAXANE Nanoparticles

Not available on PMC !

Example 1

The particles are synthesized by adding between about 5 mg and about 20 mg of rituximab (or non-specific IgG) to 20 mg of ABRAXANE. Saline is then added to a final volume of 2 ml for a final concentration of 10 mg/ml ABRAXANE, and the mixture is allowed to incubate at room temperature for 30 minutes to allow particle formation. Particles average about 160 nm and are termed “AR160” nanoparticles.

Optionally, the composition is divided into aliquots and frozen at −80° C. Once frozen the aliquots are optionally lyophilized overnight with the Virtis 3L benchtop lyophilizer (SP Scientific, Warmister, PA) with the refrigeration on. A lyophilized preparation is generated.

The dried aliquots are stored at room temperature. These samples are reconstituted in saline at room temperature for 30 minutes, followed by centrifugation for 7 minutes at 2000×g. The resulting sample is then resuspended in the appropriate buffer, as needed.

US11878061B2. Methods for improving the therapeutic index for a chemotherapeutic drug (2024-01-23). Mayo Foundation for Medical Education and Research [US]. Inventors: Svetomir N. Markovic [US], Wendy K. Nevala [US].

Patent 2024

Abraxane Albumins Buffers Centrifugation Freezing Rituximab Saline Solution

Synthesis of High-Conductivity Entropy-Stabilized Nanometal Oxide

Not available on PMC !

Example 1

Provided is a preparation method for an A-site high-entropy nanometer metal oxide (Gd_0.4Er_0.3La_0.4Nd_0.5Y_0.4)(Zr_0.7, Sn_0.8, V_0.5)O₇with high conductivity, the method including the following steps.

- (1) Gd(NO₃)₃, Er(NO₃)₃, La(NO₃)₃, Nd(NO₃)₃, Y(NO₃)₃, ZrOSO₄, SnC₁₄and NH₄VO₃were taken at a molar ratio of 0.4:0.3:0.4:0.5:0.4:0.7:0.8:0.5, added to a mixed solution of deionized water/absolute ethyl alcohol/tetrahydrofuran at a mass ratio of 0.3:3:0.5, and stirred for five minutes to obtain a mixed liquid I. The ratio of the total mass of Gd(NO₃)₃, Er(NO₃)₃, La(NO₃)₃, Nd(NO₃)₃, Y(NO₃)₃, ZrOSO₄, SnC₁₄and NH₄VO₃to that of the mixed solution of deionized water/absolute ethyl alcohol/tetrahydrofuran (0.3:3:0.5) is 12.6%.
- (2) Para-phenylene diamine, hydrogenated tallowamine, sorbitol and carbamyl ethyl acetate at a mass ratio of 1:0.2:7:0.01 were taken, added to propyl alcohol, and stirred for one hour to obtain a mixed liquid II. The ratio of the total mass of the para-phenylene diamine, the hydrogenated tallowamine, the sorbitol and the carbamyl ethyl acetate to that of the propyl alcohol is 7.5%;
- (3) The mixed liquid I obtained in step (1) was heated to 50° C., and the mixed liquid II obtained in step (2) was dripped at the speed of one drop per second, into the mixed liquid I obtained in step (1) with stirring and ultrasound, and heated to the temperature of 85° C. after the dripping is completed and the temperature was maintained for three hours while stopping stirring, and the temperature was decreased to the room temperature, so as to obtain a mixed liquid III. The mass ratio of the mixed liquid I to the mixed liquid II is 10:4.
- (4) The mixed liquid III was added to an electrolytic cell with using a platinum electrode as an electrode and applying a voltage of 3 V to two ends of the electrode, and reacting for 13 minutes, to obtain a mixed liquid IV.
- (5) The mixed liquid IV obtained in step (4) was heated with stirring, another mixed liquid II was taken and dripped into the mixed liquid IV obtained in step (4) at the speed of one drop per second. The mass ratio of the mixed liquid II to the mixed liquid IV is 1.05:1.25; and after the dripping is completed, the temperature was decreased to the room temperature under stirring, so as to obtain a mixed liquid V.
- (6) A high-speed shearing treatment was performed on the mixed liquid V obtained in step (5) by using a high-speed shear mulser at the speed of 20000 revolutions per minute for one hour, so as to obtain a mixed liquid VI.
- (7) Lyophilization treatment was performed on the mixed liquid VI to obtain a mixture I;
- (8) The mixture I obtained in step (7) and absolute ethyl alcohol were mixed at a mass ratio of 1:2 and uniformly stirred, and were sealed at a temperature of 210° C. for performing solvent thermal treatment for 18 hours. The reaction was cooled to the room temperature, the obtained powder was collected by centrifugation, washed with deionized water and absolute ethyl alcohol eight times respectively, and dried to obtain a powder I.
- (9) The powder I obtained in step (8) and ammonium persulfate was uniformly mixed at a mass ratio of 10:1, and sealed and heated to 165° C. The temperature was maintained for 13 hours. The reaction was cooled to the room temperature, the obtained mixed powder was washed with deionized water ten times, and dried to obtain a powder II.
- (10) The powder II obtained in step (4) was placed into a crucible, heated to a temperature of 1500° C. at a speed of 3° C. per minute. The temperature was maintained for 7 hours. The reaction was cooled to the room temperature, to obtain an A-site high-entropy nanometer metal oxide (Gd_0.4Er_0.3La_0.4Nd_0.5Y_0.4)(Zr_0.7, Sn_0.8, V_0.5)O₇with high conductivity.

As observed via an electron microscope, the obtained A-site high-entropy nanometer metal oxide with high conductivity is a powder, and has microstructure of a square namometer sheet with a side length of about 4 nm and a thickness of about 1 nm.

The product powder was taken and compressed by using a powder sheeter at a pressure of 550 MPa into a sheet. Conductivity of the sheet is measured by using the four-probe method, and the conductivity of the product is 2.1×10⁸S/m.

A commercially available ITO (indium tin oxide) powder is taken and compressed by using a powder sheeter at a pressure of 550 MPa into a sheet, and the conductivity of the sheet is measured by using the four-probe method.

As measured, the conductivity of the commercially available ITO (indium tin oxide) is 1.6×10⁶S/m.

US11866343B2. A-site high-entropy nanometer metal oxide with high conductivity, and preparation method thereof (2024-01-09). Zhejiang University [CN]. Inventors: Lingjie Zhang [CN], Weiwei Cai [CN], Ningzhong Bao [CN].

Patent 2024

1-Propanol 4-phenylenediamine Absolute Alcohol ammonium peroxydisulfate Cells Centrifugation Electric Conductivity Electrolytes Electron Microscopy Entropy Ethanol ethyl acetate Freeze Drying indium tin oxide Metals Molar Oxides Platinum Powder Pressure propyl acetate Solvents Sorbitol tetrahydrofuran Ultrasonography

Preparation of Cell-free Lactobacillus Fractions

Not available on PMC !

Example 1

Cell-free fractions were prepared as previously described (25). Briefly, Lactobacillus acidophilus strain La-5 was grown overnight in modified DeMann, Rogosa and Sharpe medium. (mMRS; 10 g peptone from casein, 8 g meat extract, 4 g yeast extract, 8 g D(+)-glucose, 2 g dipotassium hydrogen phosphate, 2 g di-ammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate in 1 L distilled water) (MRS; BD Diagnostic Systems, Sparks, MD). The overnight culture was diluted 1:100 in fresh medium. When the culture grew to an optical density at 600 nm (OD₆₀₀) of 1.6 (1.2×10⁸cells/ml), the cells were harvested by centrifugation at 6,000×g for 10 min at 4° C. The supernatant was sterilized by filtering through a 0.2-μm-pore-size filter (Millipore, Bioscience Division, Mississauga, ON, Canada) and will be referred to as cell-free spent medium (CFSM). Two litres of L. acidophilus La-5 CFSM was collected and freeze-dried (Unitop 600 SL, VirTis Co., Inc. Gardiner, NY., USA). The freeze-dried CFSM was reconstituted with 200 ml of 18-Ω water. The total protein content of the reconstituted CFSM was quantified using the BioRad DC protein assay kit II (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). Freeze-dried CFSM was stored at −20° C. prior to the assays.

US11857581B2. Antiviral methods and compositions comprising probiotic bacterial molecules (2024-01-02). MicroSintesis Inc. [CA]. Inventors: Mansel Griffiths [CA].

Patent 2024

ammonium citrate Biological Assay casein peptone Cells Centrifugation Diagnosis Freezing Glucose Hydrogen Lactobacillus acidophilus L Cells manganese sulfate Meat potassium phosphate, dibasic Proteins Sodium Acetate Sulfate, Magnesium Unitop Yeast, Dried

Translocation of Surface Lipoproteins in E. coli

Not available on PMC !

Example 6

TbpB and NMB0313 genes were amplified from the genome of Neisseria meningitidis serotype B strain B16B6. The LbpB gene was amplified from Neisseria meningitidis serotype B strain MC58. Full length TbpB was inserted into Multiple Cloning Site 2 of pETDuet using restriction free cloning ((F van den Ent, J. Löwe, Journal of Biochemical and Biophysical Methods (Jan. 1, 2006)).). NMB0313 was inserted into pET26, where the native signal peptide was replaced by that of pelB. Mutations and truncations were performed on these vectors using site directed mutagenesis and restriction free cloning, respectively. Pairs of vectors were transformed into E. coli C43 and were grown overnight in LB agar plates supplemented with kanamycin (50 μg/mL) and ampicillin (100 μg/mL).

tbpB genes were amplified from the genomes of M. catarrhalis strain 035E and H. influenzae strain 86-028NP and cloned into the pET52b plasmid by restriction free cloning as above. The corresponding SLAMs (M. catarrhalis SLAM 1, H. influenzae SLAM1) were inserted into pET26b also using restriction free cloning. A 6His-tag was inserted between the pelB and the mature SLAM sequences as above. Vectors were transformed into E. coli C43 as above.

Cells were harvested by centrifugation at 4000 g and were twice washed with 1 mL PBS to remove any remaining growth media. Cells were then incubated with either 0.05-0.1 mg/mL biotinylated human transferrin (Sigma-aldrich T3915-5 MG), α-TbpB (1:200 dilution from rabbit serum for M. catarrhalis and H. influenzae; 1:10000 dilution from rabbit serum for N. meningitidis), or α-LbpB (1:10000 dilution from rabbit serum-obtained a gift from J. Lemieux) or α-fHbp (1:5000 dilution from mouse, a gift from D. Granoff) for 1.5 hours at 4° C., followed by two washes with 1 mL of PBS. The cells were then incubated with R-Phycoerythrin-conjugated Streptavidin (0.5 mg/ml Cedarlane) or R-phycoerythrin conjugated Anti-rabbit IgG (Stock 0.5 mg/ml Rockland) at 25 ug/mL for 1.5 hours at 4° C. The cells were then washed with 1 mL PBS and resuspended in 200 uL fixing solution (PBS+2% formaldehyde) and left for 20 minutes. Finally, cells were washed with 2×1 mL PBS and transferred to 5 mL polystyrene FACS tubes. The PE fluorescence of each sample was measured for PE fluorescence using a Becton Dickinson FACSCalibur. The results were analyzed using FLOWJO software and were presented as mean fluorescence intensity (MFI) for each sample. For N. meningtidis experiments, all samples were compared to wildtype strains by normalizing wildtype fluorescent signals to 100%. Errors bars represent the standard error of the mean (SEM) across three experiments. Results were plotted statistically analysed using GraphPad Prism 5 software. The results shown in FIG. 6 for the SLPs, TbpB (FIG. 6A), LbpB. (FIG. 6B) and fHbp (FIG. 6C) demonstrate that SLAM effects translocation of all three SLP polypeptides in E. coli. The results shown in FIG. 10 demonstrate that translocation of TbpB from M. catarrhalis (FIG. 10C) and in H. influenzae (FIG. 10D) in E. coli require the co-expression of the required SLAM protein (Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria. Hooda Y, Lai C C, Judd A, Buckwalter C M, Shin H E, Gray-Owen S D, Moraes T F. Nat Microbiol. 2016 Feb. 29; 1:16009).

US11872275B2. SLAM polynucleotides and polypeptides and uses thereof (2024-01-16). ENGINEERED ANTIGENS INC. [CA]. Inventors: Trevor F. Moraes [CA], Christine Chieh-Lin Lai [CA], Yogesh Hooda [CA], Andrew Judd [CA], Anthony B. Schryvers [CA], Scott D. Gray-Owen [CA].

Patent 2024

ADRB2 protein, human Agar Ampicillin anti-IgG Cells Centrifugation Cloning Vectors Culture Media Escherichia coli Fluorescence Formaldehyde Genes Genome Haemophilus influenzae Homo sapiens Kanamycin Lipoproteins Membrane Proteins Moraxella catarrhalis Mus Mutagenesis, Site-Directed Mutation Neisseria Neisseria meningitidis Phycoerythrin Plasmids Polypeptides Polystyrenes prisma Rabbits Serum Signaling Lymphocytic Activation Molecule Family Member 1 Signal Peptides Strains Streptavidin Technique, Dilution Transferrin Translocation, Chromosomal Virulence Factors

Purification and Immobilization of FC4E Enzyme for Tagatose Production

Not available on PMC !

Example 9

NEBT7EL-pA06238 was grown on LB with 50 μg/ml kanamycin. A 600 ml culture of TB_kan50was inoculated with NEBT7EL-pA06238 and incubated overnight at 37° C. at 200 rpm. The next morning, a 10 L fermentor was prepared with 9.5 L of TB and then inoculated with 500 ml of the overnight culture. The culture was grown at 37° C. The pH was maintained at 6.2 with NaOH and the dO₂was maintained ≥20%. After 2 hours of growth, the temperature was dropped to 25° C. The culture was grown for an additional 1 hour with the OD₆₀₀around 7. IPTG was added to a final concentration of 1 mM and CoCl₂was added to 25 μM. Additional CoCl₂was added 1 and 2 hours after induction to bring the final concentration to 300 μM. The cells were grown for 20 hours at which point the fermentor was chilled to 10° C. and the cells were harvested by centrifugation. The cell pellet was stored at −80° C. until use.

The cell pellet from the fermentation was lysed by stirring in buffer with lysozyme and DNAse. Cell debris was removed by centrifugation and the supernatant was filtered through a 0.45 micron filter. Filtered supernatant was incubated with Ni-NTA agarose resin and then enzyme was eluted with imidazole. Purified FC4E pA06238 was immobilized onto 5.25 grams of ECR8204F resin using the standard published protocol from Purolite.

The immobilized enzyme was loaded into a 11×300 mm glass fixed bed reactor and run for approximately 200 h at constant temperature (60° C.) with a constant feed composition of 30 wt % fructose+70 wt % aqueous buffer solution (20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂). Feed rate was held constant at 140 uL/min throughout the run. The fixed bed reaction reached a maximal conversion of approximately 30% tagatose and had a half-life of −50 hours (FIG. 15).

US11866758B2. Methods and compositions for preparing tagatose from fructose (2024-01-09). Arzeda Corp. [US]. Inventors: Alexandre Zanghellini [US], Kyle Roberts [US], Michael Charles Milner Cockrem [US], Christopher Dunckley [US].

Patent 2024

ARID1A protein, human Buffers Cells Centrifugation Deoxyribonucleases Enzymes Enzymes, Immobilized Fermentation Fermentors Fructose imidazole Isopropyl Thiogalactoside Kanamycin Muramidase Resins, Plant Sepharose Sodium Chloride tagatose

Top products related to «Centrifugation»

Protease inhibitor cocktail by Roche

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Protease inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is commonly used in protein extraction and purification procedures to prevent protein degradation.

Fetal bovine serum (fbs) by Thermo Fisher Scientific

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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.

Protease inhibitor cocktail by Merck Group

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The Protease Inhibitor Cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that can degrade proteins. It is a combination of various chemical compounds that work to prevent the breakdown of proteins in biological samples, allowing for more accurate analysis and preservation of protein integrity.

Pvdf membrane by Merck Group

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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.

Trizol reagent by Thermo Fisher Scientific

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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.

Pierce bca protein assay kit by Thermo Fisher Scientific

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The Pierce BCA Protein Assay Kit is a colorimetric-based method for the quantification of total protein in a sample. It utilizes the bicinchoninic acid (BCA) reaction, where proteins reduce Cu2+ to Cu+ in an alkaline environment, and the resulting purple-colored reaction is measured spectrophotometrically.

Facscalibur by BD

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The FACSCalibur is a flow cytometry system designed for multi-parameter analysis of cells and other particles. It features a blue (488 nm) and a red (635 nm) laser for excitation of fluorescent dyes. The instrument is capable of detecting forward scatter, side scatter, and up to four fluorescent parameters simultaneously.

Trizol by Thermo Fisher Scientific

Sourced in United States, Germany, China, Japan, United Kingdom, Canada, France, Italy, Australia, Spain, Switzerland, Belgium, Denmark, Netherlands, India, Ireland, Lithuania, Singapore, Sweden, Norway, Austria, Brazil, Argentina, Hungary, Sao Tome and Principe, New Zealand, Hong Kong, Cameroon, Philippines

TRIzol is a monophasic solution of phenol and guanidine isothiocyanate that is used for the isolation of total RNA from various biological samples. It is a reagent designed to facilitate the disruption of cells and the subsequent isolation of RNA.

Complete protease inhibitor cocktail by Roche

Sourced in United States, Germany, Switzerland, United Kingdom, China, France, Japan, Canada, Spain, Belgium, Australia, Sweden, Italy, Ireland, Macao

The Complete Protease Inhibitor Cocktail is a laboratory product designed to inhibit a broad spectrum of proteases. It is a concentrated solution containing a mixture of protease inhibitors effective against a variety of protease classes. This product is intended to be used in research applications to preserve the integrity of target proteins by preventing their degradation by proteolytic enzymes.

Bca protein assay kit by Thermo Fisher Scientific

Sourced in United States, China, Germany, United Kingdom, Japan, Belgium, France, Switzerland, Italy, Canada, Australia, Sweden, Spain, Israel, Lithuania, Netherlands, Denmark, Finland, India, Singapore

The BCA Protein Assay Kit is a colorimetric detection and quantification method for total protein concentration. It utilizes bicinchoninic acid (BCA) for the colorimetric detection and quantification of total protein. The assay is based on the reduction of Cu2+ to Cu1+ by protein in an alkaline medium, with the chelation of BCA with the Cu1+ ion resulting in a purple-colored reaction product that exhibits a strong absorbance at 562 nm, which is proportional to the amount of protein present in the sample.

What are the key benefits of using centrifugation in biomedical research and clinical diagnostics?

Centrifugation is a widely used technique that allows for the purification, concentration, or isolation of various biological components, such as proteins, nucleic acids, organelles, and cells. This process enables researchers and clinicians to separate particles, cells, or molecules based on their differential sedimentation rates within a centrifugal force field, which is crucial for obtaining reliable and reproducible experimental results.

How can researchers optimize their centrifugation protocols?

Optimizing centrifugation parameters, such as speed, duration, temperature, and rotor type, is crucial for ensuring the reproducibility and reliability of experimental results. Researchers must carefully consider the specific application and sample characteristics when designing their centrifugation protocols to achieve the desired separation and purification outcomes.

What are the different types or variations of centrifugation techniques?

How can PubCompare.ai assist researchers in optimizing their centrifugation protocols?

PubCompare.ai's AI-powered platform can help researchers easily locate, compare, and identify the best centrifugation methods and products to enhance their research workflows and improve data quality. The platform's data-driven insights and recommendations can assist researchers in selecting the most effective centrifugation protocols for their specific research goals, leading to improved reproducibility and accuracy in their work.

What are some common challenges researchers face when using centrifugation, and how can PubCompare.ai address them?

Researchers may face challenges in screening large volumes of published literature to identify the most effective centrifugation protocols for their research. PubCompaer.ai's AI-driven analysis can help researchers pinpoingt critical insights, enabling them to quickly and efficiently compare different protocols and choose the best option for their needs. This can lead to improved reproducibility and data quality, as well as save researchers valuable time and resources.

More about "Centrifugation"

Centrifugation is a fundamental technique in biomedical research and clinical diagnostics, involving the separation of particles, cells, or molecules based on their differential sedimentation rates within a centrifugal force field.
This process allows for the purification, concentration, or isolation of various biological components, such as proteins, nucleic acids, organelles, and cells.
Centrifugation protocols can vary in terms of speed, duration, temperature, and rotor type, depending on the specific application and sample characteristics.
Optimizing these parameters is crucial for ensuring the reproducibility and reliability of experimental results.
Some common applications of centrifugation include protein purification using techniques like precipitation, dialysis, and column chromatography; nucleic acid extraction using reagents like TRIzol; cell fractionation for the isolation of organelles like mitochondria, lysosomes, and nuclei; and flow cytometry sample preparation on instruments like FACSCalibur.
Researchers can leverage the power of AI-driven platforms like PubCompare.ai to easily locate, compare, and identify the best centrifugation methods and products to enhance their research workflows and improve data quality.
By providing data-driven insights and recommendations, PubCompare.ai can help researchers optimize their centrifugation protocols and take their work to the next level.
Synonyms and related terms: sedimentation, ultracentrifugation, density gradient centrifugation, cell fractionation, protein purification, nucleic acid extraction, flow cytometry, protease inhibitor cocktail, fetal bovine serum (FBS), PVDF membranes, BCA protein assay kit.
Abbreviations: FBS, PVDF, BCA, FACSCalibur, TRIzol.