Effect of pore structure on protein adsorption mechanism on ion exchange media: A preliminary study using low field nuclear magnetic resonance.

The adsorption process of bovine serum albumin (BSA), ovalbumin (OVA) and human immunoglobulin G (IgG) on agarose ion-exchange media Q Sepharose FF and two dextran-grafted agarose media including Q Sepharose XL and Capto Q were studied using low field nuclear magnetic resonance (NMR). The T2 relaxation time was found directly proportional to the pore size and diminished after protein adsorbed, therefore, a theoretical model describing the relationship between protein binding amount and T2 relaxation signals was established. The model parameters, a, which reflects the contact area between the adsorbed protein and media surface, and the δ, which defined as the ratio of the protein volume to the pore volume after adsorption, were found to describe the pore occupation states of proteins in media with different pore structures very well. For small proteins, such as BSA and OVA, monolayer adsorption occurred on Q Sepharose FF, which has no dextran chains. Therefore, the adsorbed protein only occupied 49.05% of the pore volume for BSA and 25.51% for OVA, and contact area of each protein on the media were also low, suggesting mostly monolayer adsorption occurred. In the contrast, their adsorption to Q Sepharose XL and Capto Q with dextran chains tended to form multilayer adsorption, thus higher contact area was obtained and the pore volumes were almost 100% occupied. For large protein, such as IgG, the adsorption to all these three media was similar and about 30% of the pore volume were occupied, probably due to the similar restriction for IgG to entering the media pore. Results of this study will help to elucidate the relationship between protein adsorption and pore size variation, which present the significance of low field NMR in understanding protein adsorption mechanism.

Construction of polymer monolithic columns in polypropylene ink-pen tubes for separation of proteins by cation-exchange chromatography.

We describe the synthesis of polymer monoliths inside polypropylene tubes from ink pens. These tubes are cheap, chemically stable, and resistant to pressure. UV-initiated grafting with 5 wt% benzophenone in methanol for 20 min activated the internal surface, thus enabling the covalent binding of ethylene glycol dimethacrylate, also via photografting. The pendant vinyl groups attached a poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith prepared via photopolymerization. These tubes measured 100-110 mm long, with 2 mm of internal diameter. The parent monoliths were functionalized with Na2 SO3 or iminodiacetate to produce strong and weak cation exchangers, respectively. The columns exhibited permeabilities varying from 2.7 to 3.3 × 10-13  m2 , which enabled the separation of proteins at 500 µL/min and back pressures <2.8 MPa. Neither structure collapse nor monolith detachment occurred at flow rates as high as 2.0 mL/min, which produced back pressures between 6.9 and 9.0 MPa. The retention times of ovalbumin, ribonuclease A, cytochrome C, and lysozyme in salt gradient at pH 7.0 followed the order of increasing isoelectric points, confirming the cation exchange mechanism. Separation and determination of lysozyme in egg white proved the applicability of the columns to the analysis of complex samples.

Fast construction of polymer monolithic columns inside fluorinated ethylene propylene (FEP) tubes for separation of proteins by reversed-phase liquid chromatography.

This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm2. ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10-13 m2, providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 µL min-1, collapsing only at flow rates >700 µL min-1, a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples.

An easy and rapid separation method for five major proteins from egg white: Successive extraction and MALDI-TOF-MS identification.

Five major proteins from egg white were separated using a successive extraction/precipitation protocol. The yield and purity of the separated proteins were measured. The separated proteins were confirmed by MALDI-TOF-MS, and their structures were characterized by CD spectrum. Lysozyme was first separated using FPC 3500 resin and then ovomucin from the lysozyme-free egg white. Ammonium sulfate and citric acid were added to the resulting lysozyme- and ovomucin-free egg white solution to precipitate ovotransferrin. Ovomucoid and ovalbumin were separated from the resulting supernatant using ethanol. The separated proteins were further purified and the optimal conditions for the further purifications were suggested. The purity and yield of lysozyme, ovotransferrin, ovalbumin, and ovomucoid were higher than 90% and 77%, while those of ovomucin were about 72% and 75%, respectively. This study separated five major proteins in egg white successively using resin adsorption, pH adjustment, salt/ethanol precipitation, and ultrafiltration.

A facile and general approach for the preparation of boronic acid-functionalized magnetic nanoparticles for the selective enrichment of glycoproteins.

Biomedical applications and biomarkers for early clinical diagnostics and the treatment of diseases demand efficient and selective enrichment platforms for glycoproteins. Thus, we herein report a facile and general approach for the preparation of boronic acid-functionalized magnetic nanoparticles for the selective enrichment of glycoproteins. More specifically, Fe3O4 magnetic nanoparticles were initially prepared via a solvothermal reaction, and core-shell-structured Fe3O4@SiO2 nanoparticles were obtained according to a sol-gel process. Subsequently, the Fe3O4@SiO2 surfaces were modified using 4-formylphenylboronic acid to allow the formation of strong yet reversible covalent bonds between boronic acid (BA) and the cis-1,2-diol groups of glycoproteins. The morphology and structure of the Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2-BA nanoparticles were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and vibrating sample magnetometry, thereby confirming their successful preparation. The obtained BA-modified Fe3O4@SiO2 magnetic nanoparticles were applied in the attempted enrichment of two glycoproteins (ovalbumin (OVA) and transferrin (TRF)) and two non-glycoproteins (bovine serum albumin (BSA) and cytochrome c (Cyt C)). The results confirmed a significant difference in affinity between glycoproteins and non-glycoproteins. In addition, the recognition capability of the Fe3O4@SiO2-BA nanoparticles was demonstrated by the selective enrichment of glycoproteins from a complex system containing both glycoproteins (i.e., TRF) and non-glycoproteins (i.e., Cyt C).

Large-scale purification of ovalbumin using polyethylene glycol precipitation and isoelectric precipitation.

In addition to small amounts of minerals and carbohydrates, most of the dry matter of chicken egg white is protein, making egg white an ideal resource for obtaining food proteins. Ovalbumin (OVA), which accounts for more than 50% of the total egg white protein, is one of the most widely studied food proteins due to its multiple functional properties, and it has also been used as a model protein molecule in many research fields. The objective of this study was to develop a simple and rapid method for the purification of OVA from egg whites on large scale. First, OVA was separated from ovomucin, ovotransferrin, and ovomucoid by polyethylene glycol (PEG) concentration, using the following optimal parameters: the PEG concentration was 15%, the pH was 6.5, the salt concentration was 100 mmol/L, and the operating temperature was 10°C. The OVA-rich supernatant obtained from PEG precipitation was further purified by isoelectric precipitation at a pI of 4.5 and a temperature of 4°C, and the purified OVA was obtained with at a purity of 95.1% by HPLC, with a yield of 46.4%. After the extraction of OVA, the PEG solution was vacuum dried and then utilized cyclically in the PEG precipitation steps. The whole purification process could be finished within 2 to 3 h at a scale of several kilograms of egg white. This method has the advantages of rapidity, simplicity, low cost, and ease of scalability.

Preparation of ionic liquid modified magnetic metal-organic frameworks composites for the solid-phase extraction of α-chymotrypsin.

A novel magnetic solid-phase extraction (MSPE) method based on 1-hexyl-3-methyl imidazolium chloride ionic liquid (IL) modified magnetic Fe3O4 nanoparticles, hydroxylated multiwall carbon nanotubes (MWCNTs-OH) and zeolitic imidazolate frameworks (ZIFs) nanocomposites (Fe3O4-MWCNTs-OH@ZIF-67@IL) were proposed and applied to extract α-chymotrypsin. The magnetic materials were synthesized successfully and characterized by X-ray diffraction (XRD), transmission electron microscope (TEM), thermal gravimetric analysis (TGA), fourier transform infrared spectrometry (FT-IR), vibrating sample magnetometer (VSM) and zeta potentials. Subsequently, the UV-vis spectrophotometer at about 280 nm was utilized to quantitatively analyze the α-chymotrypsin concentration in the supernatant. Furthermore, single factor experiments revealed that the extraction capacity was influenced by initial α-chymotrypsin concentration, ionic strength, extraction time, extraction temperature and pH value. The extraction capacity could reach up to about 635 mg g-1 under the optimized conditions, absolutely higher than that of extraction for Ovalbumin (OVA), Bovine serum albumin (BSA) and Bovine hemoglobin (BHb). In addition, the regeneration studies showed Fe3O4-MWCNTs-OH@ZIF-67@IL particles could be reused several times and kept a high extraction capacity. Besides, the study of enzymatic activity also indicated that the activity of the extracted α-chymotrypsin was well maintained 93% of initial activity. What's more, the proposed method was successfully applied to extract α-chymotrypsin in porcine pancreas crude extract with satisfactory results. All of above conclusions highlight the great potential of the proposed Fe3O4-MWCNTs-OH@ZIF-67@IL-MSPE method in the analysis of biomolecules.

Allowance for radial dilution in evaluating the concentration dependence of sedimentation coefficients for globular proteins.

The accuracy with which the concentration dependence of the sedimentation coefficient, s = s 0(1 - kc), can be quantified for globular proteins by commonly used procedures has been examined by subjecting simulated sedimentation velocity distributions for ovalbumin to c(s)‒s analysis. Because this procedure, as well as its g(s)‒s counterpart, is based on assumed constancy of s over the time course of sedimentation coefficient measurement in a given experiment, the best definition of the concentration coefficient k is obtained by associating the measured s with the mean of plateau concentrations for the initial and final distributions used for its determination. The return of a slightly underestimated k (by about 3%) is traced to minor mislocation of the air‒liquid meniscus position as the result of assuming time independence of s in a given experiment. Although more accurate quantification should result from later SEDFIT and SEDANAL programs incorporating the simultaneous evaluation of s 0 and k, the procedures based on assumed constancy of s suffice for determining the limiting sedimentation coefficient s 0-the objective of most s‒c dependence studies.

Practical aspects of the automated preparation of aqueous two phase systems for the analysis of biological macromolecules.

A robust strategy for the automated preparation of aqueous two-phase systems (ATPS) using a liquid handling sample processor was developed using gravimetric methods: to determine the accuracy of preparation. The major robotic control parameters requiring adjustment were; speed of aspiration and dispense; delay times following aspiration and dispense alongside measures to control cross-contamination during phase sampling. In general mixture compositions of both polymer/polymer and polymer/salt mixtures could be prepared with a target bias accuracy of less than 5%. However, we found that the bias accuracy with which systems of defined TLL and MR could be constructed was highly dependent on the tie line length of the ATPS and the geometrical form of the ATPS co-existence curve. For systems with a very low degree of curvature (PEG/salt systems here) increases in bias (accuracy) are appreciable at relatively long tie line lengths. Where the degree of curvature is more pronounced (PEG/dextran systems) closer approach to the critical point was possible without major effect on bias/accuracy. Application of the strategy to the measurement of the partitioning of phosphorylated and dephosphorylated forms of the model protein ovalbumin are reported. Differences in partition of phosphorylated (native) forms and dephosphorylated forms could be demonstrated. In a PEG/salt system this was manifest as a substantial decrease in solubility based on overall protein recovery derived from accurate knowledge of the system mass ratio. In a PEG/dextran system differences in partition coefficient could be demonstrated between phosphorylated and dephosphorylated forms.

Functionalized gold nanoparticles as affinity nanoprobes for multiple lectins.

Glycan-lectin interactions are commonly observed in nature. Analytical methods, which are used to detect lectins that rely on the use of glycan ligand-modified nanoprobes as affinity probes, have been developed. Most of the existing methods are focused on the use of synthetic glycan ligands. Nevertheless, naturally available glycoproteins, such as ovalbumin in chicken egg whites, are good sources for fabricating glycan-immobilized nanoprobes. In this study, we generated functionalized gold nanoparticles (Au NPs) from a one-pot reaction by reacting chicken egg white (cew) proteins with aqueous tetrachloroaurate. The generated Au@cew NPs are mainly encapsulated by ovalbumin, in which the surface is decorated by abundant hybrid mannose and Galß(1→4)GlcNAc-terminated glycan ligands. Thus, the generated Au@cew NPs containing hybrid mannose and Galß(1→4)GlcNAc have the capability to selectively bind with their corresponding lectins. Lectins including concanavalin A, banana lectin, and ricin B that have binding moieties toward specific sugars were used as the model samples. Our results showed that the generated AuNPs can be used as multiplex affinity probes for these model lectins. Lectins can be selectively released from the Au@cew NP-lectin conjugates by using specific sugars, such as mannose, glucose, and ß-lactose, as the releasing agents to release specific lectins from the conjugates. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used as the tool to characterize the released species from the nanoprobes. The limit of detection of these model lectins using the current approach was low (in nM). The feasibility of using the Au@cew NP-based affinity MALDI-MS to selectively detect specific lectins from complex samples was also demonstrated.

Comparison of Different Methods of Purification and Concentration in Production of Influenza Vaccine.

The overwhelming majority of influenza vaccines are prepared with the use of chicken embryo allantoic fluid. The presence of ovalbumin (this protein constitutes >60% total protein in the allantoic fluid) in the vaccine can lead to severe allergy. Hence, effective reduction of ovalbumin content is of crucial importance for vaccine production. We compared two methods of purification and concentration of influenza virus: zonal gradient ultracentrifugation and combined ultrafiltration/diafiltration and exclusion chromatography protocol, used for fabrication of seasonal vaccines. Combined chromatography is comparable with zonal centrifugation protocol by the results of ovalbumin removal (to meet standard requirements).

Fabrication of an Open Microfluidic Device for Immunoblotting.

Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an "enclosed" microchannel for PAGE and blotting to an "open" PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.

Synthesis of hydrophilic and conductive molecularly imprinted polyaniline particles for the sensitive and selective protein detection.

In this work, a novel kind of water-dispersible molecular imprinted conductive polyaniline particles was prepared through a facile and efficient macromolecular co-assembly of polyaniline with amphiphilic copolymer, and applied as the molecular recognition element to construct protein electrochemical sensor. In our strategy, an amphiphilic copolymer P(AMPS-co-St) was first synthesized using 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and styrene (St) as monomer, which could co-assemble with PANI in aqueous solution to generate PANI particles driven by the electrostatic interaction. During this process, ovalbumin (OVA) as template protein was added and trapped into the PANI NPs particles owing to their interactions, resulting in the formation of molecular imprinted polyaniline (MIP-PANI) particles. When utilizing the MIP-PANI particles as recognition element, the resultant imprinted PANI sensor not only exhibited good selectivity toward template protein (the imprinting factor α is 5.31), but also a wide linear range over OVA concentration from 10-11 to 10-6mgmL-1 with a significantly lower detection limit of 10-12mgmL-1, which outperformed most of reported OVA detecting methods. In addition, an ultrafast response time of less than 3min has also been demonstrated. The superior performance is ascribed to the water compatibility, large specific surface area of PANI particles and the electrical conductivity of PANI which provides a direct path for the conduction of electrons from the imprinting sites to the electrode surface. The outstanding sensing performance combined with its facile, quick, green preparation procedure as well as low production cost makes the MIP-PANI particles attractive in specific protein recognition and sensing.

Fabrication of diverse pH-sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples.

In proteomic studies, poor detection of low abundant proteins is a major problem due to the presence of highly abundant proteins. Therefore, the specific removal or depletion of highly abundant proteins prior to analysis is necessary. In response to this problem, a series of pH-sensitive functional mesoporous silica materials composed of 2-(diethylamino)ethyl methacrylate and methacrylic acid units were designed and synthesized via atom transfer radical polymerization. These functional mesoporous silica materials were characterized and their ability for adsorption and separation of proteins was evaluated. Possessing a pH-sensitive feature, the synthesized functional materials showed selective adsorption of some proteins in aqueous or buffer solutions at certain pH values. The specific removal of a particular protein from a mixed protein solution was subsequently studied. The analytical results confirmed that all the target proteins (bovine serum albumin, ovalbumin, and lysozyme) can be removed by the proposed materials from a five-protein mixture in a single operation. Finally, the practical application of this approach was also evaluated by the selective removal of certain proteins from real biological samples. The results revealed that the maximum removal efficiencies of ovalbumin and lysozyme from egg white sample were obtained as 99% and 92%, respectively, while the maximum removal efficiency of human serum albumin from human serum sample was about 80% by the proposed method. It suggested that this treatment process reduced the complexity of real biological samples and facilitated the identification of hidden proteins in chromatograms.

Polydopamine assisted fabrication of titanium oxide nanoparticles modified column for proteins separation by capillary electrochromatography.

Development of a simple method for preparation of stable open tubular (OT) columns for proteins separation by capillary electrochromatography is still challenging. In this work, the titanium oxide (TiO2) nanoparticles coated OT column was successfully prepared for separation of proteins by capillary electrochromatography. The polydopamine (PDA) film was first formed in the inner surface of a fused-silica capillary by the self-polymerization of dopamine under alkaline conditions. Then the TiO2 coating was deposited onto the surface of pre-modified capillary with PDA by a liquid phase deposition process. The plentifully active hydroxyl groups in PDA coating can chelate with Ti(4+) to boost the nucleation and growth of TiO2 film. The as-prepared TiO2 coated OT column was characterized by scanning electron microscopy and measurement of electroosmotic flow. Furthermore, the influence of liquid phase deposition time on the TiO2 coating was investigated. The TiO2 coated OT column was used for successful separation of two variants of ß-lactoglobulin and eight glycoisoforms of ovalbumin. The column demonstrated good repeatability and stability. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.7%. Moreover, the application of the column was verified by successful separation of acidic proteins in egg white.

Preparation of a novel Zr(4+)-immobilized metal affinity membrane for selective adsorption of phosphoprotein.

In this study, a novel phosphate-Zr(4+) immobilized metal affinity membrane (IMAM) was prepared based on the surface initiated-atom transfer radical polymerization technique for the selective adsorption of phosphoprotein. The adsorption capacity and selectivity of the phosphate-Zr(4+) IMAM were evaluated by using the mixture of standard phosphoproteins (ß-casein, ovalbumin) and nonphosphoproteins (bovine serum albumin and lysozyme) as model samples. The adsorption isotherms and competitive adsorption results demonstrated that the phosphate-Zr(4+) IMAM had higher binding capacity and selectivity for phosphoproteins over nonphosphoproteins. Moreover, the phosphate-Zr(4+) IMAM exhibited good re-usability and re-productivity. Finally, the phosphate-Zr(4+) IMAM was applied to separate phosphoprotein from real samples with high purity. Therefore, the as-prepared phosphate-Zr(4+) IMAM could be a promising affinity material for the efficient enrichment of phosphoprotein from complex bio-samples.

Purification of recombinant ovalbumin from inclusion bodies of Escherichia coli.

Recombinant ovalbumin expressed in bacterial host is essentially free from post-translational modifications and can be useful in understanding the structure-function relationship of the protein. In this study, ovalbumin was expressed in Escherichia coli in the form of inclusion bodies. Ovalbumin inclusion bodies were solubilized using urea and refolded by decreasing the urea concentration by dilution. Refolded protein was purified by anion exchange chromatography. Overall recovery of purified recombinant ovalbumin from inclusion bodies was about 30% with 98% purity. Purified recombinant ovalbumin was characterized by mass spectrometry, circular dichroism and fluorescence spectroscopy. Recombinant ovalbumin was shown to be resistant to trypsin using protease resistance assay. This indicated proper refolding of ovalbumin from inclusion bodies of E. coli. This method provides a simple way of producing ovalbumin free of post-translational modifications.

Impact of extraction and elution media on non-size effects in size exclusion chromatography of proteins.

Size exclusion chromatography is extensively used to separate proteins and to determine their apparent molecular weights. It separates proteins based on hydrodynamic volume, but interactions between the chromatography resin and proteins lead to non-size effects. This report discusses the impact of co-solvents [salt, urea, sodium dodecyl sulfate (SDS), dithiothreitol] in extraction media when separating wheat gluten proteins, soy glycinin, bovine serum albumin and ovalbumin on a Biosep-SEC-S4000 column. With acetonitrile/water (1:1, v/v) containing 0.05% (v/v) trifluoroacetic acid as eluent, salts and SDS in the extraction media increase while urea decreases non-size effects. Most gluten and globular proteins are extractable in sodium phosphate buffer (0.050M; pH 6.8) containing 2.0% (w/v) SDS. This chromatographic medium allows analyzing mixtures of various proteins without any non-size effects.

Carboxyl modified magnetic nanoparticles coated open tubular column for capillary electrochromatographic separation of biomolecules.

Carboxyl modified magnetic nanoparticles (Fe3O4-COOH MNPs) coated open tubular (OT) columns were prepared for capillary electrochromatography. The Fe3O4-COOH MNPs coatings were constructed on the surface of positively charged poly(diallydimethylammonium chloride) (PDDA) modified capillaries through electrostatic self-assembly approach. The as-prepared PDDA@Fe3O4-COOH MNPs coated OT columns were characterized by scanning electron microscopy and electroosmotic flow measurement. The electrochromatographic characterization of the OT columns was evaluated by separation of amino acids, dipeptides and proteins. The influences of background solution pH, concentration, and organic modifier content on separation were investigated. The separation of these analytes was primarily based on the electrophoretic mechanism in combination with chromatographic mechanism. The Fe3O4-COOH MNPs coatings improved the separation resolution of these analytes due to their large surface area. Three variants of bovine serum albumin, two variants of ß-lactoglobulin and nine glycoisoforms of ovalbumin were successfully separated. The relative standard deviations of migration times of analytes representing run-to-run, day-to-day and column-to-column were less than 4.3%. Furthermore, the feasibility of the PDDA@Fe3O4-COOH MNPs coated OT column was verified by successful separation of acidic proteins in egg white.

Different components of chicken ovalbumin extract promote different cell proliferation.

The chicken ovalbumin extracts could promote cell survival and proliferation. In the present study, the different components in chicken ovalbumin extracts were further separated to find the component primarily responsible for promoting cell proliferation. Components of differing molecular weight were separated from chicken ovalbumin extracts by ultrafiltration. Different components were co—cultured with different cells at different final concentrations, and the effects on cell proliferation were subsequently determined by a CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega). Components from chicken ovalbumin extracts less than 3 kD in size can promote 293T cell and 293T—GFP cell proliferation. The components from chicken ovalbumin extract more than 3 kD in size can promote bone marrow or umbilical cord mesenchymal stem cell (MSC) proliferation. The separation of components from chicken ovalbumin less than and more than 3 kD in size that are able to function as active components for the promotion of different cellular proliferation. This discovery may identify a new and convenient additive for cell culture media, promoting cell growth and proliferation.