Log In Sign up
The largest database of trusted experimental protocols
> Chemicals & Drugs > Amino Acid > Plasma protein fraction

Plasma protein fraction

Plasma protein fraction is a complex mixture of proteins derived from human or animal plasma.
These fractions are used in various medical and research applications, such as the treatment of immunodeficiencies, bleeding disorders, and other conditions.
PubCompare.ai helps optimize plasma protein fraction research by enabling users to easily locate and compare protocols from literature, pre-prints, and patents.
Leveraging AI-driven comparisons, PubCompare.ai enhances reproducibility and accuracy, ensuring researchers find the best protocols and products for their studies.
Boost your plasma protein fraction reasearch with this innovative tool.

Most cited protocols related to «Plasma protein fraction»

1
Subcellular Fractionation of Tissue and Cells
Cited 65 times
This method modifies a previously published protocol based on cultured cells [1] (link), expanding it to allow for the collection of subcellular fractions from fresh tissue. In addition, the buffer compositions (Table 1) were optimized to minimize nuclear protein loss via the addition of 1 M hexylene glycol, which helps to further stabilize the membranes, especially that of the nucleus, and has been previously shown to yield highly enriched nuclear fractions [2] (link). HEPES is an organic buffer that stabilizes the pH of the solution while NaCl maintains the ionic strength [2] (link).
The basis of this method (Fig. 1) is the sequential lysis of cell membranes by increasing the detergent strength of lysis buffers to obtain proteins from each fraction. Lysis buffer A is meant to release cytosolic proteins and its main component is digitonin. Digitonin is a steroidal saponin that permeabilizes the plasma membrane by binding with cholesterol and other β-hydroxysterols, thereby leading to the formation of pores in the membrane and its subsequent disruption. The advantage of lysing cells with digitonin is that it is unable to disrupt the membranes of cellular organelles, as the cholesterol composition of these membranes is lower [3] (link). Lysis buffer B releases the proteins from all membrane bound organelles except the nucleus. The main component of this buffer is igepal, which is a non-ionic, non-denaturing detergent chemically equivalent to Nonidet P-40 [4] (link). It is used at a low concentration to allow permeabilization of the endoplasmic reticulum, Golgi and mitochondria membranes, while keeping the nuclear membrane intact. Lysis buffer C is meant to permeabilize the nuclear membrane and release the nuclear proteins. Among its components, lysis buffer C contains sodium deoxycholate, a mild, non-ionic and non-denaturing biological detergent (a constituent of bile). Sodium dodecyl sulphate is an anionic detergent that is extremely effective in membrane solubilization [5] . The combination of sodium deoxycholate and sodium dodecyl sulphate thus creates an effective nuclear lysis buffer. Benzonase is added prior to the isolation of nuclear fractions and digests DNA and RNA, facilitating the complete release of all nuclear proteins [1] (link).
Equipment required for this method:

End-over-end tube rotator

Handheld homogenizer (rotor/stator type)

Qiagen QIAshredder columns (Qiagen, 79656)

Microcentrifuge

Fractionation protocol for isolated tissue (Fig. 1):

Mince fresh, unfrozen tissue into 2–4 mm pieces, wash with 1 mL of ice cold phosphate buffered saline solution (10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

Add 40–60 mg of tissue into a 1.5 mL microtube.

Add 500 μL of ice cold lysis buffer A supplemented with 5 μL protease inhibitor cocktail.

Disrupt the tissue for 5 s using a hand held tissue homogenizer (rotor/stator type).

Transfer the tissue suspension to QIAshredder homogenizer (Qiagen, 79656) and centrifuge at 500 × g for 10 min at 4 °C to filter the homogenate.

Discard the top filter containing trapped tissue particles, and resuspend the pellet in the filtrate by gently pipetting up and down. Transfer into a new tube.

Add 500 μL of ice cold lysis buffer A (1.5 mL if working with brain tissue), and supplement with 5 μL of protease inhibitor cocktail.

Incubate the homogenate on an end-over-end rotator for 10 min at 4 °C

Centrifuge at 4000 × g for 10 min at 4 °C.

Collect the supernatant. This fraction contains the cytosolic proteins.

Using a 1 mL pipette and tip, resuspend the pellet by gently pipetting up and down in 1 mL of ice cold lysis buffer B supplemented with 10 μL of protease inhibitor cocktail. Incubate for 30 min on an end-over-end rotator at 4 °C.

Centrifuge at 6000 × g for 10 min at 4 °C.

Collect the supernatant. This fraction contains the proteins from membrane-bound organelles (mitochondria, endoplasmic reticulum, Golgi, etc.) except those from the nucleus.

Add 500 units of benzonase (Sigma, E1014) to 20 μL of water and combine it with the pellet from step 12.

Resuspend the pellet by gently flicking the bottom of the tube and incubate at room temperature for 15 min.

Add 500 μL of ice cold lysis buffer C with 5 μL of protease inhibitor cocktail to the benzonase-digested pellet and incubate on an end-over-end rotator for 10 min at 4 °C.

Pellet the insoluble material by centrifuging at 6800 × g for 10 min at 4 °C.

Collect the supernatant. This fraction contains the nuclear proteins.

Pellet contains nuclear proteins and protein complexes that resist extraction and typically include active RNA polymerases and regulatory proteins. These can be solubilized with lysis buffer C supplemented with 8 M urea for analysis, or discarded.

Fractionation protocol for cultured cells (Fig. 1):The following protocol is optimized for cultured cells grown on a 100 mm diameter dish (55 cm2 surface area).

Remove culture medium and wash the cells with room temperature phosphate buffered saline solution.

Trypsinize the cells by adding 800 μL of 0.25% Trypsin: 0.9 mM EDTA: phenol red solution (Gibco Life Technologies, 25200) and incubating the cells at 37 °C for 2 min or until cells are detached.

Add 5 mL of culture medium containing 10% fetal bovine serum to inhibit trypsin activity, and collect the cells.

Centrifuge at 500 × g for 10 min at 4 °C to pellet the cells.

Using a 1 mL pipette and tip, discard the supernatant and resuspend the pellet by pipetting up and down in 500 μL of ice cold PBS.

Centrifuge for at 500 × g for 10 min at 4 °C to pellet the cells.

Discard the supernatant and add 400 μL of ice cold lysis buffer A supplemented with 4 μL of protease inhibitor cocktail.

Incubate on end-over-end rotator for 10 min at 4 °C.

Centrifuge at 2000 × g for 10 min at 4 °C.

Collect the supernatant. This fraction contains the cytosolic proteins.

Add 400 μL of ice cold lysis buffer B supplemented with 4 μL of protease inhibitor cocktail and resuspend the pellet by vortexing.

Incubate on ice (or at 4 °C) for 30 min.

Centrifuge at 7000 × g for 10 min at 4 °C.

Collect the supernatant. This fraction contains the proteins from membrane-bound organelles (mitochondria, endoplasmic reticulum, Golgi, etc.) except those from the nucleus.

Add 400 μL of ice cold lysis buffer C containing 7 μL of Benzonase and 4 μL of protease inhibitor cocktail.

Incubate on an end-over-end rotator for 30 min at 4 °C to allow complete solubilization of nuclei and digestion of genomic DNA.

Centrifuge at 7800 × g for 10 min at 4 °C.

Collect the supernatant. This fraction contains the nuclear proteins.

Pellet contains nuclear proteins and protein complexes that resist extraction and typically include active RNA polymerases and regulatory proteins. These can be solubilized with lysis buffer C supplemented with 8 M urea for analysis, or discarded.

Using the technique developed for fractionation of isolated tissue, freshly isolated rat hearts perfused free of blood with Krebs-Henseleit solution at 37 °C using the Langendorff technique [6] (link), were fractionated into cytosolic (C), membrane bound organelle (M) and nuclear (N) fractions. The purity of the fractions was assessed by western blotting against specific markers (Fig. 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, antibody 14C10, Cell Signaling Technology, Beverly, MA, USA) was used as the major cytosolic marker [7] (link). The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2, antibody ab137020, Abcam, Cambridge, UK) and voltage-dependent anion channel (VDAC, antibody ab15895, Abcam) were used as membrane markers since they are associated with the sarco/endoplasmic reticulum and mitochondria, respectively [8] (link). Lamin A/C (antibody 2032S, Cell Signaling Technology, Beverly, MA, USA) a major structural protein of the nuclear membrane, was used as a nuclear marker [1] (link).
GAPDH can be found in the mitochondria and small vesicular structures of the cell when exposed to stressors which cause a dynamic subcellular redistribution of GAPDH [7] (link). Consistent with this, while the bulk of GAPDH is found in the cytosolic fraction, a much smaller amount is present in the membrane fraction. GAPDH is not seen in the nuclear fraction of hearts (Fig. 2A) or human fibrosarcoma HT1080 cells (Fig. 2B), confirming nuclear fraction purity. Both SERCA2 and VDAC were present in the membrane fraction from hearts, but absent from the cytosolic and nuclear fractions (Fig. 2A). Lamin A/C was found exclusively in the nuclear fractions isolated from hearts (Fig. 2A) and primarily in the nuclear fraction isolated from HT1080 cells, with substantially less appearing in the membrane fraction (Fig. 2B). The presence of the nuclear membrane-associated protein lamin A/C mostly in the nuclear fraction indicates that the nuclear membrane is contained in this fraction. These results confirm the purity of the fractions collected from tissue and cells with the protocol presented above.
Baghirova S., Hughes B.G., Hendzel M.J, & Schulz R. (2015). Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells. MethodsX, 2, 440-445.
Publication 2015
2
Optimizing Circulating miRNA Analysis
Cited 56 times
Plasma and serum are characterized by the presence of RNases and a low yield of RNA, which indeed cannot be measured by spectrophotometric determination. While microarray- and sequence-based approaches are currently available [25 (link)], qPCR has been the preferred one for the quantification of circulating miRNAs, given its sensitivity and specificity and high dynamic range [26 (link)]. High-throughput qPCR platforms exist for assaying multiple miRNAs in parallel, either with stem-loop reverse transcription (RT) reaction combined with TaqMan qPCR, or with a poly(A)-tailed RT combined with SYBR Green detection and LNA primers [26 (link), 27 (link)]. The major limitation of this approach is that it cannot identify novel miRNAs, but for human studies, this factor can be less problematic, given the fact that human miRNA repertoire is well defined.
In the bloodstream, miRNAs have been shown to reside within a vesicle-associated fraction, including exosomes, microvescicles and apoptotic bodies [28 (link)] or protected by multi-protein complexes [7 (link)]. Although circulating miRNAs are consistently reported to be stable in serum after long-term storage and treatment with RNases or freeze/thaw cycles [1 (link), 2 (link), 6 (link)], they are influenced by compartmentalization, as vesicle-associated miRNAs seem to be more stable than those present in a non-membrane-bound form [29 (link), 30 (link)]. Therefore, the measured variability is a result of true biological changes, which are associated with the phenomenon or disease of interest, and of technical, nonspecific variability, which is introduced during the multiple experimental steps. Potential sources of technical variability include: (a) the starting material and collection/isolation procedures (serum, plasma, exosomes, other vesicles); (b) multiple freeze-thaw cycles; (c) the RNA extraction method and its efficiency; (d) the input RNA quantity and quality; (e) the efficiency of the enzymatic reactions; and (f) the inter-individual variation in total RNA and miRNA concentration in plasma or serum. Therefore, the accuracy of the reported associations is dependent on a careful study design and appropriate data normalization. To ensure that proper measures are taken to minimize confounding factors, below we go through some considerations to guide the study design for the identification of reference circulating miRNAs.

Keep homogeneous conditions. When using archived samples, select samples that have been processed according to the same standardized protocol, to reduce technical variation from this initial step in the data. For example, make sure to avoid hemolyzed serum specimens, as cellular RNA will contaminate the serum fraction and prevent the accurate determination of circulating miRNA profiles [31 (link), 32 (link)]. Although plasma and isolated vesicles from blood represent an alternative starting material, mixing different collection methods should be obviously avoided, because the availability of circulating miRNAs in different biological compartments and distinctive efficiency and specificity will result in different miRNA identification and quantification [33 , 34 (link)]. To further reduce experimentally induced variability, RNA extraction method must be thoroughly tested and standardized, as the yield of Trizol-based or column-based methods can result in qualitative and quantitative differences [35 (link), 36 (link)].

Reduce freeze/thaw cycles. Given that the ‘packaging’ of circulating miRNAs might influence their stability, it is possible that repeated freeze/thaw cycles of serum samples would differentially affect their levels [30 (link)] and negatively influence the identification of both stable and differentially expressed miRNAs.

Reduce batch effects. A batch may be defined as a subgroup of samples or experiments, exhibiting a systematic nonbiological difference that is not correlated with the biological variables under study (for example, different experiment days, laboratory conditions, reagent lots or operators). Batch effects represent a common source of variability in high-throughput methods and should be avoided or reduced. Although methods exist [37–39 (link)] to reduce such unwanted variation, those methods fail when a biological variable is totally confounded with a batch variable. Nevertheless, when the experimental goal is to find normalizers, it is amenable not to mix different experimental batches.

Add a synthetic RNA for technical normalization. The efficiency of RNA extraction, complementary DNA synthesis and PCR amplification can be monitored using an exogenous synthetic miRNA (for example, Caenorhabditis elegans cel-miR-39 or Arabidopsis thaliana ath-miR-159a), which are added as spike-in control during processing. When added to the lysis buffer during the extraction step, the synthetic RNA will be put through the same conditions as the endogenous miRNAs, hence providing a process control, for technical normalization [1 (link)]. However, this type of external reference will not be able to correct for other sources of variability, as for example the total concentration of the miRNA fraction in serum, which is likely changing inter-individually and/or in a disease-associated fashion. For a comprehensive normalization protocol, endogenous controls are required, either using global high-throughput-derived parameters (i.e. mean expression value per sample) or selected, validated stable reference miRNAs for focused assays. While miR-16 has been used to normalize the results in many studies, this miRNA was shown to be particularly susceptible to hemolysis, thus being far from an ideal choice as an internal control [22 (link)].

Marabita F., de Candia P., Torri A., Tegnér J., Abrignani S, & Rossi R.L. (2015). Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR. Briefings in Bioinformatics, 17(2), 204-212.
Publication 2015
3
Comparative Evaluation of PBPK Models
Cited 37 times
The four models in httk include: “pbtk”, “3compartment”, “3compartmentss”, and “1compartment”; the predictions and parameters of these models are compared in Table 2. All models currently use only oral and intravenous (i.v.) dosing. The models pbtk and 3compartment, shown in Figure 1, use tissue to unbound plasma partition coefficients calculated with a modified version of Schmitt’s model (Schmitt 2008b (link); Pearce et al. 2017 ) (using octanol-water partitioning, membrane affinity, acid/base dissociation constants, tissue compositions, and adjusted fub) to simulate chemical concentrations over time for multiple tissue compartments. The model pbtk contains separate tissue compartments for the gut, liver, lungs, arteries, veins, and kidneys while the model 3compartment only contains compartments for the liver and gut and is essentially a condensed form of the model pbtk. The tissues contained in tissue.data that are unused in each of these models are aggregated into a single compartment termed “rest”, whose partition coefficient is calculated by averaging the remaining partition coefficients, weighted by their species-specific tissue volumes. Absorption from the gut lumen into gut tissue is modeled as a first order process with an arbitrary “fast” absorption rate of 1 h−1. The fraction of the dose absorbed into the system through the gut wall is set to 1 when measured data are unavailable. The gut blood flows directly into the liver, where the hepatic clearance, Clmetabolism, is calculated with a unit conversion of Clint using the density of hepatocytes in the liver (1.1 × 108 hepatocytes per gram of liver from Ito and Houston 2004 (link) and a liver density of 1.05 g/mL from Snyder et al. 1975 ). Both models also feature renal elimination by passive glomerular filtration through the kidneys. We assume perfusion-limited tissue (i.e., tissue, red blood cells, and plasma come to equilibrium rapidly with respect to the flow of blood), and a constant Rblood2plasma is used throughout the body, predicted using hematocrit and the predicted partitioning between red blood cells and plasma when in vivo values are unavailable.
The models 3compartmentss and 1compartment both contain only plasma without separate compartments for blood and tissue (and thus no individual partition coefficients). The model 3compartmentss, “ss” standing for steady state, is a single equation for the Css of the rest-of-body compartment in the model 3compartment resulting from i.v. dosing. This is the same equation used for determining Css in previous work (Rotroff et al. 2010 (link); Wetmore et al. 2012 (link); Wetmore 2015 (link); Wilkinson and Shand 1975 (link)) but with a modification adjusting for the misuse of hepatic blood flow in determining plasma clearance (Yang et al. 2007 (link)). The model 1compartment features an absorption compartment and a total clearance equal to the sum of the metabolism of the parent compound in the liver, modeled with the adjusted “well-stirred” approximation (Wilkinson and Shand 1975 (link); Houston and Carlile 1997 (link)), and the renal clearance by passive glomerular filtration. The elimination rate, ke, is equal to the total clearance divided by the volume of distribution, Vdist. Vdist is used as the volume of the compartment and is calculated by summing the plasma volume and the products of each tissue to unbound plasma partition coefficient, its corresponding volume, and fub (Schmitt 2008b (link)). Css resulting from infusion dosing for the model 1compartment is equivalent to 3compartmentss.
Among the four models in the package, the simplest model, 3compartmentss, is applicable to the largest number of chemicals, specifically those which are missing information needed to parameterize the other models. It is the only model that does not use partition coefficients and thus does not require logP, and when fub is below the limit of detection, the model can be used with Monte Carlo to simulate Css distributions. Thus, fub below the limit of detection (set to zero in chem.physical_and_invitro.data and 0.005 in default parameter lists) and Clint are the minimum data requirements for running a model. The model 1compartment is included to compare our predictions with in vivo experiments which are often characterized by one compartment model parameters (Vdist and kelim). We note that fully understanding the kinetics of a given chemical might require additional data on features currently not accessible with high-throughput in vitro approaches, such as bioavailability, transporters, protein-binding kinetics, and extra-hepatic or strongly saturable metabolism (Rotroff et al. 2010 (link)).
Pearce R.G., Setzer R.W., Strope C.L., Wambaugh J.F, & Sipes N.S. (2017). httk: R Package for High-Throughput Toxicokinetics. Journal of statistical software, 79(4), 1-26.
Publication 2017
4
SARS-CoV-2 Spike Pseudotyped Lentivirus Neutralization Assay
Cited 19 times
Neutralization assays were conducted using pseudotyped lentiviral particles, as described elsewhere [31 ], with a few modifications. First, we used a spike protein with a cytoplasmic tail truncation that removes the last 21 amino acids (spike ∆21). The map for this plasmid, HDM-SARS2-Spike-delta21, is in Supplementary File 1, and the plasmid is available from Addgene (plasmid no. 155130). We used a spike protein with a C-terminal deletion because, since publication of our original protocol [31 ], other groups have reported that deleting the spike protein’s cytoplasmic tail improves titers of spike-pseudotyped viruses [32–35 (link)]. Indeed, we found that the C-terminal deletion increased the titers of our pseudotyped lentiviral particles without affecting neutralization sensitivity (Supplementary Figure 2).
For our neutralization assays, we seeded black-walled, clear bottom, poly-L-lysine coated 96-well plates (Greiner; no. 655936) with 1.25 × 104 293T-ACE2 (NR-52511) cells per well in 50 μL of D10 medium (Dulbecco modified Eagle medium with 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) at 37ºC with 5% carbon dioxide. About 12 hours later, we diluted the plasma samples in D10, starting with a 1:20 dilution followed by 6 or 11 serial 3-fold dilutions (11 dilutions were used for samples from individuals in whom we had previously measured high neutralizing antibody responses; PIDs 13, 23, and 25). We then diluted the spike-∆21 pseudotyped lentiviral particles 1:6 (1 mL of virus plus 5 mL of D10 per plate) and added a volume of virus equal to the volume of plasma dilution to each well of the plasma dilution plates. We incubated the virus and plasma for 1 hour at 37ºC and then added 100 μL of the virus-plus-plasma dilutions to the cells.
At 50–52 hours after infection, luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega; E2610) as described elsewhere [31 ], except that luciferase activity was measured directly in the assay plates. Two “no-plasma” wells were included in each row of the neutralization plate, and the fraction infectivity was calculated by dividing the luciferase readings from the wells with plasma by the average of the no-plasma wells in the same row. After calculating fraction infectivities, we used the neutcurve Python package (https://jbloomlab.github.io/neutcurve/) to calculate the plasma dilution that inhibited infection by 50% (IC50), fitting a Hill curve with the bottom fixed at 0 and the top at 1. The NT50 for each plasma sample was calculated as the reciprocal of the IC50. Individuals whose plasma was not sufficiently neutralizing to interpolate an IC50 using the Hill curve fit were assigned an NT50 of 20 (the limit of our dilution series) for plotting (Figures 1A, 1C, and 2B) and for fold-change analyses (Figure 1B).
All samples were assayed at least in duplicate. We analyzed all samples from the same individual in the same batch of neutralization assays and on the same plate when possible. Each batch of samples included a negative control of pooled serum samples collected from 2017–2018 (Gemini Biosciences; nos. 100–110, lot H86W03J; pooled from 75 donors), and 1 plasma sample known to be neutralizing (from PID 4C at the 30-day time point). These samples were used to confirm consistency between batches.
Results from SARS-CoV-2 spike-pseudotyped lentivirus neutralization assays have been shown to correlate well with full virus SARS-CoV-2 neutralization assays [36 (link), 37 (link)]. Nonetheless, in an effort to help standardize comparisons between neutralization assays, we also performed our assay with a standard serum sample from the National Institute for Biological Standards and Control (NIBSC) (Research Reagent for Anti-SARS-CoV-2 Ab; NIBSC code 20/130). This sample had an NT50 of approximately 3050 (Supplementary Figure 3).
Crawford K.H., Dingens A.S., Eguia R., Wolf C.R., Wilcox N., Logue J.K., Shuey K., Casto A.M., Fiala B., Wrenn S., Pettie D., King N.P., Greninger A.L., Chu H.Y, & Bloom J.D. (2020). Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection. The Journal of Infectious Diseases, 223(2), 197-205.
Publication 2020
5
Plasma Proteome Depletion and Analysis
Cited 17 times
Human blood collection and plasma processing were performed using the protocol developed by the Clinical Proteomics Technologies Assessment for Cancer (CPTAC) Biospecimen Working Group (http://proteomics.cancer.gov/). The 14 most abundant proteins were depleted from batches of 40 μL of human plasma using a MARS-Hu-14 (4.6 × 100 mm) column from Agilent (Santa Clara, CA) on a Michrom Paradigm MG4 HPLC system (Auburn, CA) following the manufacturer's instructions. The protein flow-through fraction was collected and readjusted to the original volume using a 5 kDa MWCO centrifugal concentrator (Sartorius AG, Goettingen, Germany). Depleted plasma samples were reduced with DTT, alkylated with IAM, and digested using trypsin as previously described.25 (link) One microliter aliquots of peptide mixture (approximately 1 μg/μL) were analyzed using an Eksigent nano-LC 2D HPLC system (Eksigent, Dublin, CA) connected to a quadrupole time-of-flight (QqTOF) QSTAR Elite mass spectrometer (MDS SCIEX, Concorde, Canada). Peptides were loaded on a guard column (C18 Acclaim PepMap100, 300 μm i.d. × 5 mm, 5 μm particle size, 100 Å pore size, Dionex, Sunnyvale, CA), washed for 10 min using mobile phase A containing 0.1% FA at a flow rate of 20 μL/min, and transferred to an analytical C18-nanocapillary HPLC column (C18 Acclaim PepMap100, 300 μm i.d. × 15 cm, 3 μm particle size, 100 Å pore size, Dionex, Sunnyvale, CA). Mobile phase B consisted of ACN with 0.1% FA. Peptides were separated using a gradient of 2–40% B for 120 min, followed by a rapid increase of B from 40–90% in 15 min, and held at 90% B for 9 min before returning to initial conditions of 98% A (flow rate 300 nL/min). All mass spectra were recorded at a resolution of 12 000–15 000. Following each survey scan, the six most abundant ions were selected and fragmented using the advanced information dependent acquisition (IDA) feature along with QSTAR Elite specific features such as “Smart Collision” and “Smart Exit” (fragment intensity multiplier set to 2.0 and maximum accumulation time of 1.5 s). MS/MS spectra were acquired using the dynamic exclusion feature (exclusion mass width 50 mDa m/z and exclusion duration of 60 s) of the mass spectrometer. A total of five replicate LC-MS/MS experiments were performed and 35 290 MS/MS spectra were collected. WIFF files were processed using Protein Pilot software (version 2.0.1, Applied Biosystems, Carlsbad, CA) and tandem mass spectra were exported in MGF file format. Generated MGF files were transcoded to mzXML format using LibMSR software, a precursor of the ProteoWizard24 (link) library.
Ma Z.Q., Dasari S., Chambers M.C., Litton M.D., Sobecki S.M., Zimmerman L.J., Halvey P.J., Schilling B., Drake P.M., Gibson B.W, & Tabb D.L. (2009). IDPicker 2.0: Improved Protein Assembly with High Discrimination Peptide Identification Filtering. Journal of proteome research, 8(8), 3872-3881.
Publication 2009
A-A-1 antibiotic ARID1A protein, human BLOOD cDNA Library DNA Replication High-Performance Liquid Chromatographies Homo sapiens Ions Malignant Neoplasms Mass Spectrometry Peptides Plasma Proteins Radionuclide Imaging Tandem Mass Spectrometry Technology Assessment Trypsin

Most recents protocols related to «Plasma protein fraction»

1
Quantifying Plasma Radiometabolites for PET Tracers
Not available on PMC !
Arterial blood samples were drawn at 0, 5, 10, 20, 40, 60 and 90 min p.i. and the plasma was separated by centrifugation (4°C, 2118 g, 5 min). The plasma proteins were precipitated by adding 700 µL of acetonitrile to 500 µL of plasma, vortexing and centrifuging (3370 g, 3 min). The protein free supernatant was analyzed with high-performance liquid chromatography (HPLC) using a method described in the supplementary material of Brumberg et al. [25] to obtain fractions of intact [ 11 C]SMW139 and its radioactive metabolites for correcting the plasma TAC. A radioactive standard was prepared by spiking the time point 0 plasma supernatant with [ 11 C]SMW139 in order to analyze the correct peak of the chromatograms to correspond to the parent.
Parent and radiometabolite binding to plasma proteins was analyzed for a subset of subjects from blood samples drawn prior to [ 11 C]SMW139 injection and from 20 min p.i. From the time point 0 plasma drawn for in vitro protein binding analysis, 1 mL was frozen for later duplicate analysis. The in vitro plasma and in vivo 20 min parent fraction analysis plasma samples were used to analyze parent and radiometabolite plasma protein binding with separate ultra ltration membrane corrections.
Lehto J., Aarnio R., Tuisku J., Sucksdorff M., Koivumäki E.M., Nylund M., Helin S., Rajander J., Danon J.J., Gilchrist J., Kassiou M., Oikonen V., & Airas L. (2024). P2X7-receptor binding in new-onset and secondary progressive MS – a [11C]SMW139 PET study.
Publication 2024
2
Membrane Protein Extraction Protocol
For membrane protein preparation, samples were homogenized on ice with a plasma membrane protein extraction kit (Invent Biotechnologies, USA, SM-005). The efficiency of membrane protein extraction has been demonstrated in our previous work [41 (link)]. The detailed protocol was as follows: DRG tissues (L4–L6) were lysed with buffer A. The filter cartridge was capped and centrifuged at 16,000 × g for 30 s. The filter was discarded, and the pellet was resuspended and centrifuged at 700 × g for 1 min (the pellet contained the intact nuclei). The supernatant was transferred to a new tube and centrifuged for 10–30 min at 16,000 × g. The supernatant (the cytosolic fraction) was removed, and the pellet (the total membrane protein fraction including organelles and plasma membranes) was saved. The total membrane protein fraction was resuspended in buffer B and centrifuged at 7800 × g for 5 min. The pellet contained the organelle membrane proteins (in our study, cytoplasmic protein comprised the cytosolic fraction and organelle membrane fraction). The supernatant was carefully transferred to a fresh 2.0 ml microcentrifuge tube, and 1.6 ml ice-cold PBS was added. The sample was mixed a few times by inverting and centrifuged at 16,000 × g for 15–30 min. The supernatant was discarded, the pellet (isolated plasma membrane proteins) was saved, and the BCA method was used to determine the protein concentration. Protein samples of different fractions were denatured and prepared for immunoblotting.
Xie M.X., Lai R.C., Xiao Y.B., Zhang X., Cao X.Y., Tian X.Y., Chen A.N., Chen Z.Y., Cao Y., Li X, & Zhang X.L. (2024). Endophilin A2 controls touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking. Military Medical Research, 11, 17.
Publication 2024
3
Lipoprotein Fractionation in Quail
Not available on PMC !
Plasma TC (Siedel et al. 1983) ,TG (Ziegenhorn, 1975) , 91 Beef tallow diets and aortic plaque in quail (Bradford, 1976) were determined using standard methods (Boehringer Mannheim). Lipoproteins were separated from whole plasma by density gradient ultracentrifugation (Terpstra et al. 1982) . Plasma lipoprotein fractions in samples from each treatment were visualized by running a reference tube pre-stained with Sudan black in ethylene glycol. Sucrose density gradient measurements and Sudan black staining of lipoprotein fractions in individual treatment reference samples were used to confirm the density gradients used in unstained samples. Quail fed on the highcholesterol (HC) diets exhibited a distinct lipid plug on the top (r 20 Ͻ1 . 006) of the centrifuge tube after ultracentrifugation which corresponded to the portomicron fraction. This fraction was carefully removed to prevent contamination of other fractions before their removal (Terpstra et al. 1982) . Remaining lipoproteins were separated into five fractions at density ranges as follows: fraction 1, 1 . 006 Ͻ r 20 Ͻ1 . 020; fraction 2, 1 . 030 Ͻ r 20 Ͻ 1 . 046; fraction 3, 1 . 050 Ͻ r 20 Ͻ 1 . 080; fraction 4, 1 . 106 Ͻ r 20 Ͻ 1 . 184; fraction 5, r 20 Ͼ 1 . 21 using an SW 40Ti rotor at 272 000 g for 22 h at 20Њ in a Beckman L2-65 ultracentrifuge (Beckman, Montreal, Quebec, Canada). Lipoprotein fractions were assayed for lipids and protein as previously noted for whole plasma. Preliminary analysis of lipoprotein fractions for protein content indicated that the density ranges chosen for individual lipoprotein fractions allowed the recovery of equivalent fractions from plasma of birds fed on low-cholesterol and high-cholesterol diets (Fig. 1).
Yuan Y., Kitts D.D., & Godin D.V. (2024). Interactive effects of increased intake of saturated fat and cholesterol on atherosclerosis in the Japanese quail.
Publication 2024
4
Estimating Unbound Drug Concentration
Peak plasma concentrations (Cmax, μM = ng/mL/MW, where MW is the molecular weight of the compound) and the unbound fraction of most drugs were obtained from the literature, or from FDA-approved package inserts. The unbound fraction of apilimod (not found in the literature) was estimated based on chemical structure, using a PK computer program, which uses a random forest machine learning method (DruMAP ver. 1.5, Mizhguchi Laboratory, Tokyo, Japan, https://drumap.nibiohn.go.jp/, Accessed 5 April 2024) [87 (link)]. Drugs that exhibited binding to plasma proteins were assumed to equilibrate rapidly between the protein-bound and unbound fractions in the plasma, and that only the unbound fraction was free to interact with cells [88 (link)]. The unbound concentration of drug in the plasma at Cmax (unbound Cmax) was calculated by multiplying Cmax by the unbound fraction of drug.
Hurwitz S.J., De R., LeCher J.C., Downs-Bowen J.A., Goh S.L., Zandi K., McBrayer T., Amblard F., Patel D., Kohler J.J., Bhasin M., Dobosh B.S., Sukhatme V., Tirouvanziam R.M, & Schinazi R.F. (2024). Why Certain Repurposed Drugs Are Unlikely to Be Effective Antivirals to Treat SARS-CoV-2 Infections. Viruses, 16(4), 651.
Publication 2024
5
Measuring ADH and ALDH2 Activities
ADH activity was measured in cytosol of mouse and human liver, in the cytosolic fraction of AML-12 cells and in peripheral serum and plasma, respectively, using the method previously described by Ming et al. [19 (link)]. Acetaldehyde dehydrogenase 2 (ALDH2) activity in the mitochondrial fraction of mouse and human livers was determined using a commercial kit following the protocol of the manufacturer (#E4587; BioVision, MA, USA). For the isolation of mitochondrial and cytosolic fraction liver tissue was homogenized in 1.15 % ice cold potassium chloride and fractionated by ultracentrifugation (100 000 g for 1 h). Cells were homogenized and sonicated for 5 min and then centrifuged by ultracentrifugation (43 000 g for 1 h) for isolation of the cytosolic fraction. The enzymatic kinetic measurement for ADH activity was immediately conducted and changes in extinction at 340 nm for 1 h at 37 °C were measured. All measurements in the cytosolic and mitochondrial fraction of liver tissue were normalized to the cell number (determined with DNA concentrations by measuring absorbance (260/280 nm) in the nuclear fraction of the cells), in the cytosolic fraction of AML-12 cells to the whole protein concentration (determined with Bradford assay) and in serum/plasma to the amount of ADH1 protein determined by Western Blot (see below).
Burger K., Jung F., Staufer K., Ladurner R., Trauner M., Baumann A., Brandt A, & Bergheim I. (2024). MASLD is related to impaired alcohol dehydrogenase (ADH) activity and elevated blood ethanol levels: Role of TNFα and JNK. Redox Biology, 71, 103121.
Publication 2024

Top products related to «Plasma protein fraction»

1
Whatman by Cytiva
Whatman is a brand of laboratory filtration products and equipment manufactured by Cytiva. Whatman products are designed for a variety of laboratory applications, including sample preparation, purification, and analysis. The product line includes filter papers, membranes, and related accessories.
2
Hitrap by Cytiva
The HiTrap is a line of ready-to-use prepacked chromatography columns designed for fast and convenient purification of proteins and other biomolecules. The columns are prepacked with a variety of different chromatography media, allowing users to select the appropriate separation technique for their specific application.
3
Hybond by Cytiva
Hybond is a family of nylon-based membranes used for the transfer and immobilization of biomolecules, such as proteins and nucleic acids, in various blotting techniques. These membranes provide a stable and efficient platform for the capture and detection of target analytes.
4
Biacore t200 by Cytiva
Sourced in United States, Sweden, New Zealand, United Kingdom
The Biacore T200 is a label-free interaction analysis system designed for real-time monitoring and quantification of biomolecular interactions. It provides kinetic and affinity data to support decision-making in drug discovery and development processes.
5
Mabselect by Cytiva
Mabselect is a protein A affinity chromatography resin designed for the purification of monoclonal antibodies (mAbs) and Fc-containing proteins. It provides high dynamic binding capacity and purity for efficient antibody capture and purification.
6
Plasma membrane protein extraction kit by Abcam
Sourced in United States, United Kingdom
The Plasma Membrane Protein Extraction Kit is designed for the isolation of plasma membrane proteins from various cell types. It utilizes a series of centrifugation steps to fractionate cellular components and enrich for plasma membrane proteins.
7
Amersham ecl by Cytiva
Sourced in Japan, United States
The Amersham ECL is a chemiluminescent detection system used for the identification and quantification of proteins in Western blotting applications. It provides a sensitive and reliable method for detecting low-abundance proteins.
8
Las 4000 by Cytiva
Sourced in United States
The LAS-4000 is a compact, high-performance imaging system designed for life science applications. It provides sensitive detection and quantification of proteins, nucleic acids, and other biological samples using chemiluminescence and fluorescence detection methods.
9
Hiprep by Cytiva
HiPrep is a preparative chromatography column used for purification of biomolecules. It is designed for fast and efficient separation of proteins, peptides, nucleic acids, and other biomolecules. The column is pre-packed with a variety of media, allowing for a wide range of applications.
10
Predictor by Cytiva
The Predictor is a laboratory instrument designed for automated sample processing and analysis. The core function of the Predictor is to streamline and standardize sample handling and preparation workflows. It is capable of performing various liquid handling tasks such as pipetting, mixing, and dilution to ensure consistent and reliable results.

More about "Plasma protein fraction"

Plasma protein fraction (PPF) is a complex mixture of proteins derived from human or animal plasma.
These protein fractions are widely used in various medical and research applications, such as the treatment of immunodeficiencies, bleeding disorders, and other conditions.
PPF can also be referred to as blood plasma proteins, blood plasma protein fractions, or plasma protein concentrates.
The fractionation and purification of plasma proteins are commonly performed using techniques like chromatography, filtration, and precipitation.
Researchers may utilize tools like Whatman filter papers, Hitrap affinity columns, Hybond membranes, and Biacore T200 biosensors to isolate and analyze specific plasma protein components.
To further study and characterize plasma proteins, researchers often employ methods like Western blotting (using Amersham ECL detection systems and LAS-4000 imaging devices) and affinity purification (utilizing Mabselect or Hiprep chromatography columns).
The Plasma Membrane Protein Extraction Kit can also be used to extract and enrich plasma membrane proteins from biological samples.
Leveraging AI-driven comparison tools like PubCompare.ai can help optimize plasma protein fraction research by enabling users to easily locate and compare protocols from literature, pre-prints, and patents.
This innovative tool enhances reproducibility and accuracy, ensuring researchers find the best protocols and products for their studies.
Boost your plasma protein fraction reasearch with PubCompare.ai and stay ahead of the curve in this exciting field of biomedical research.