Partition Efficiency of Eccentric Coil for Countercurrent Chromatographic Separation of Proteins Using Small-scale Cross-axis Coil Planet Centrifuge with Circular and Elliptic Cylindrical Columns.

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

Bovine serum albumin and myoglobin separation by size exclusion SMB.

The separation of the proteins Bovine Serum Albumin (BSA) and Myoglobin (Mb) was achieved by Size-Exclusion Simulated Moving Bed (SE-SMB) and performed experimentally in the FlexSMB® unit, an SMB unit designed and built in the Laboratory of Separation and Reaction Engineering. Before accomplishing the separation experiments in the mentioned unit, separation regions were computed by simulation based on a phenomenological mathematical model to determine appropriate operating conditions. The developed model was validated in advance, against fixed-bed dynamic adsorption experimental results, for pure component and binary mixtures. Then the SMB experiments were carried out, and purities of the Mb on the extract and BSA on the raffinate streams were 98% and 96%, respectively. The achieved recoveries were 80% of Mb on the extract and 94% of BSA on the raffinate. Lastly, productivities of 6.4 gprotein⋅lads-1⋅day-1 for the extract and 28.8 gprotein⋅lads-1⋅day-1 for the raffinate were obtained.

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.

Genetic and functional diversity of the multiple lungfish myoglobins.

It is known that the West African lungfish (Protopterus annectens) harbours multiple myoglobin (Mb) genes that are differentially expressed in various tissues and that the Mbs differ in their abilities to confer tolerance towards hypoxia. Here, we show that other lungfish species (Protopterus dolloi, Protopterus aethiopicus and Lepidosiren paradoxa) display a similar diversity of Mb genes and have orthologous Mb genes. To investigate the functional diversification of these genes, we studied the structures, O2 binding properties and nitrite reductase enzymatic activities of recombinantly expressed P. annectens Mbs (PanMbs). CD spectroscopy and small-angle X-ray scattering revealed the typical globin-fold in all investigated recombinant Mbs, indicating a conserved structure. The highest O2 affinity was measured for PanMb2 (P50  = 0.88 Torr at 20 °C), which is mainly expressed in the brain, whereas the muscle-specific PanMb1 has the lowest O2 affinity (P50  = 3.78 Torr at 20 °C), suggesting that tissue-specific O2 requirements have resulted in the emergence of distinct Mb types. Two of the mainly neuronally expressed Mbs (PanMb3 and PanMb4b) have the highest nitrite reductase rates. These data show different O2 binding and enzymatic properties of lungfish Mbs, reflecting multiple subfunctionalisation and neofunctionalisation events that occurred early in the evolution of lungfish. Some Mbs may have also taken over the functions of neuroglobin and cytoglobin, which are widely expressed in vertebrates but appear to be missing in lungfish.

Development of A Lateral Flow Highway: Ultra-Rapid Multitracking Immunosensor for Cardiac Markers.

The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.

Identification of chicken meat quality via rapid array isoelectric focusing with extraction of hemoglobin and myoglobin in meat sample.

Isoelectric focusing (IEF) has been used for determination of meat quality with high stability analysis. However, it still suffered from time-consuming, laborious and cost-effective performances, e.g., 3 h protein extraction, more than 10 h rehydration time, 5-12 h focusing time, and imaging of protein band. To overcome these issues, a speedy extraction of colorful proteins was developed by controlling extraction and centrifugation of 0.2g sample within 10 min and 15 min respectively; a rapid analytical method was designed by using a quick array IEF with 25 min rehydration, 7 min focusing, 2 min online scanning and imaging of focused proteins. The total analytical time was well controlled within 1 h, significantly less than the traditional IEF time of 24 h. To demonstrate the proposed method, 18 chickens were classified into three groups, e.g., the normal slaughtering, death treatment underwater, and death with infection via the New castle disease (NDV) virus. The experiments demonstrated that two Mb bands with pI 6.8 and 7.4 were present in slaughtered chickens, while four other bands with pI 6.83, 6.95, 7.09, and 7.13 were observed in abnormal chicken. The additional four proteins bands were identified by western blot (WB) as hemoglobin proteins. Furthermore, array Immobilized pH Gradient (IPG) has high sensitivity (absolute LOD of Mb and Hb were 1.3 ng and 5.5 ng), fair stability (RSD values of 2.32%, 2.27%, and 1.69%) for slaughtered, drowned, NDV-infected chickens for intra-day and (2.94%, 1.66%, and 1.07%) for inter-days, and good recovery (100%, 98.25% and 99.75%). Finally, the developed method could be used for the identification of chicken meat quality with less time and small volume reagents consuming.

Online Comprehensive High pH Reversed Phase × Low pH Reversed Phase Approach for Two-Dimensional Separations of Intact Proteins in Top-Down Proteomics.

A proof-of-concept study is presented on the use of comprehensive two-dimensional liquid chromatography mass spectrometry (LC × LC-MS) for the separation of intact protein mixtures using a different mobile phase pH in each dimension. This system utilizes mass spectrometry (MS) friendly pH modifiers for the online coupling of high pH reversed phase liquid chromatography (HPH-RPLC) in the first dimension (1D) followed by low pH reversed phase liquid chromatography (LPH-RPLC) in the second dimension (2D). Owing to the ionic nature of proteins, the use of a different mobile phase pH was successful to provide altered selectivity between the two dimensions, even for closely related protein variants, such as bovine cytochrome c and equine cytochrome c, which differ by only three amino acids. Subminute gradient separation of proteins in the second dimension was successful to minimize analysis time, while maintaining high peak capacity. Unlike peptides, the elution order of studied proteins did not follow their isoelectric points, where acidic proteins would be expected to be more retained at low pH (and basic proteins at high pH). The steep elution isotherms (on-off retention mechanism) of proteins and the very steep gradients utilized in the second-dimension column succeeded in overcoming pH and organic solvent content mismatch. The utility of the system was demonstrated with a mixture of protein standards and an Escherichia coli protein mixture.

Isoelectric focusing on microfluidic paper-based chips.

Isoelectric focusing (IEF), a powerful technique for protein separation and enrichment, was successfully integrated into microfluidic paper-based analytical devices (µPADs) in this work. The µPADs for isoelectric focusing were fabricated by octadecyltrichlorosilane (OTS) silanization and subsequent region-selective plasma treatment. The system of IEF on µPADs could be easily assembled. And a series of conditions of the system were investigated, including the suitable concentration of ampholyte to create good pH gradient, the effect of polyvinylpyrrolidone (PVP) on electroosmotic flow (EOF) suppression, and focusing voltage applied on the paper channel. After optimization, simultaneous separation and enrichment of protein sample containing myoglobin and cytochrome C was successfully demonstrated. Besides, parallel IEF on multichannels were also achieved for the separation of multiple protein samples on one single chip, and their performance was compared with that of the conventional gel-IEF system. The developed IEF on µPADs exhibits appealing features such as low cost, simplicity, and disposability and are believed to have great application potentials.

Muskox myoglobin: purification, characterization and kinetics studies compared with cattle and water buffalo myoglobins.

BACKGROUND: The Arctic muskox has economic potential as an alternative meat species and is becoming increasingly popular. The present study aimed to determine the primary structure and pseudoperoxidase activity of muskox myoglobin (Mb) compared to cattle and water buffalo myoglobins. RESULTS: The primary structure of muskox Mb was determined via a matrix-assisted laser desorption ionization-time of flight mass spectrometry-based mapping approach using the sheep Mb as a reference sequence. The muskox Mb consists of 153 amino acid residues and shows 100% identity with sheep Mb, whereas 98.69% and 97.38% identity is found with cattle and water buffalo Mbs, respectively. Muskox Mb has an autoxidation rate (MetMb formation) higher than both cattle and water buffalo Mbs at pH 7.2 (37 °C). Moreover, its pseudoperoxidase activity is higher than both cattle and water buffalo Mbs at pH 7.4 (physiological pH), whereas it is slightly lower than cattle Mb and higher than water buffalo at a lower pH (5.8), corresponding to the conditions in meat. CONCLUSION: For the first time, the present study reports the purification of myoglobin from muskoxen and, furthermore, a comparative study is conducted on autoxidation and pseudoperoxidase activity with respect to cattle and water buffalo Mbs at both physiological and acid pH. Overall, the results of the current research provide novel information for future studies useful to the meat industry when considering the importance of myoglobin as a principal pigment in meat colour stability. © 2019 Society of Chemical Industry.

High-permeability alternatives to current dialyzers performing both high-flux hemodialysis and postdilution online hemodiafiltration.

Most high-flux dialyzers can be used in both hemodialysis (HD) and online hemodiafiltration (OL-HDF). However, some of these dialyzers have higher permeability and should not be prescribed for OL-HDF to avoid high albumin losses. The aim of this study was to compare the safety and efficacy of a currently used dialyzer in HD and OL-HDF with those of several other high permeability dialyzers which should only be used in HD. A prospective, single-center study was carried out in 21 patients. Each patient underwent 5 dialysis sessions with routine dialysis parameters: 2 sessions with Helixone (HD and postdilution OL-HDF) and 1 session each with steam sterilized polyphenylene, polymethylmethacrylate (PMMA), and medium cut-off (MCO) dialyzers in HD treatment. The removal ratios (RR) of urea, creatinine, ß2 -microglobulin, myoglobin, prolactin, α1 -microglobulin, α1 -acid glycoprotein, and albumin were compared intraindividually. A proportional part of the dialysate was collected to quantify the loss of various solutes, including albumin. Urea and creatinine RRs with the Helixone-HDF and MCO dialyzers were higher than with the other 3 dialyzers in HD. The ß2 -microglobulin, myoglobin and prolactin RRs with Helixone-HDF treatment were significantly higher than those obtained with all 4 dialyzers in HD treatment. The ß2 -microglobulin value obtained with the MCO dialyzer was also higher than that obtained with the other 3 dialyzers in HD treatment. The myoglobin RR with MCO was higher than those obtained with Helixone and PMMA in HD treatment. The prolactin RR with Helixone-HD was significantly lower than those obtained in the other 4 study sessions. The α1 -microglobulin and α1 - acid glycoprotein RRs with Helixone-HDF were significantly higher than those obtained with Helixone and PMMA in HD treatment. The albumin loss varied from 0.54 g with Helixone-HD to 3.3 g with polyphenylene. The global removal score values ((UreaRR + ß2 -microglobulinRR + myoglobinRR + prolactinRR + α1 -microglobulinRR + α1 -acid glycoproteinRR - albuminRR )/6) were 43.7% with Helixone-HD, 47.7% with PMMA, 54% with polyphenylene, 54.8% with MCO and 59.6% with Helixone-HDF, with significant differences. In conclusion, this study confirms the superiority of OL-HDF over HD with the high-flux dialyzers that allow both treatments. Although new dialyzers with high permeability can only be used in HD, they are in an intermediate position and some are very close to OL-HDF.

Medium Cut-Off Dialyzer versus Eight Hemodiafiltration Dialyzers: Comparison Using a Global Removal Score.

BACKGROUND: A novel class of membranes, medium cut-off (MCO) membranes, has recently been designed to achieve interesting removal capacities for middle and large middle molecules in hemodialysis (HD) treatments. The few studies published to date have reported contradictory results regarding middle-sized molecules when comparing MCO dialyzers versus dialyzers used in online hemodiafiltration (OL-HDF). METHODS: A prospective, single-center study was carried out in 22 patients. Each patient underwent 9 dialysis sessions with routine dialysis parameters, one with an MCO dialyzer in HD and the other 8 with different dialyzers in OL-HDF. The removal ratio (RR) of urea, creatinine, ß2-microglobulin, myoglobin, prolactin, α1-microglobulin, α1-acid glycoprotein, and albumin was intraindividually compared. Albumin loss in dialysate was measured. We propose a global removal score ([ureaRR + ß2-microglobulinRR + myoglobinRR + prolactinRR + α1-microglobulinRR + α1-acid glycoproteinRR]/6 - albuminRR) as a new tool for measuring dialyzer effectiveness. RESULTS: No significant differences in the RRs of small and middle molecular range molecules were observed between the MCO vs. OL-HDF dialyzers (range 60-80%). Lower RRs were found for α1-microglobulin and α1-acid glycoprotein without significant differences. The albumin RR was < 11% and dialysate albumin loss was < 3.5 g in all situations without significant differences. The global removal score was 54.9 ± 4.8% with the MCO dialyzer without significant differences. CONCLUSIONS: Removal of a wide range of molecular weights, calculated with the proposed global removal score, was almost equal with the MCO dialyzer in HD treatment compared with 8 high-flux dialyzers in high-volume OL-HDF without relevant changes in albumin loss. The global removal score could be a new tool to evaluate the effectiveness of dialyzers and/or different treatment modalities.

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States.

Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor differences in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo- and apo-myoglobin (Mb) and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted 2H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with 2H during separation based on their disulfide-dependent sizes. The resulting differences in deuteration pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of the heme group by comparing the deuteration patterns of holo- and apo-Mb.

A Multi-Region Magnetoimpedance-Based Bio-Analytical System for Ultrasensitive Simultaneous Determination of Cardiac Biomarkers Myoglobin and C-Reactive Protein.

Cardiac biomarkers (CBs) are substances that appear in the blood when the heart is damaged or stressed. Measurements of the level of CBs can be used in course of diagnostics or monitoring the state of the health of group risk persons. A multi-region bio-analytical system (MRBAS) based on magnetoimpedance (MI) changes was proposed for ultrasensitive simultaneous detection of CBs myoglobin (Mb) and C-reactive protein (CRP). The microfluidic device was designed and developed using standard microfabrication techniques for their usage in different regions, which were pre-modified with specific antibody for specified detection. Mb and CRP antigens labels attached to commercial Dynabeads with selected concentrations were trapped in different detection regions. The MI response of the triple sensitive element was carefully evaluated in initial state and in the presence of biomarkers. The results showed that the MI-based bio-sensing system had high selectivity and sensitivity for detection of CBs. Compared with the control region, ultrasensitive detections of CRP and Mb were accomplished with the detection limits of 1.0 pg/mL and 0.1 pg/mL, respectively. The linear detection range contained low concentration detection area and high concentration detection area, which were 1 pg/mL⁻10 ng/mL, 10⁻100 ng/mL for CRP, and 0.1 pg/mL⁻1 ng/mL, 1 n/mL⁻80 ng/mL for Mb. The measurement technique presented here provides a new methodology for multi-target biomolecules rapid testing.

Macromolecular crowding induces molten globule state in the native myoglobin at physiological pH.

Here, we report the formation of molten globule state of the native myoglobin in crowded environment. We have used Soret absorption spectroscopy and far-UV circular dichroism to monitor changes in tertiary and secondary structures of myoglobin, respectively. Our results reveal that in the presence of ficoll 70, the secondary structure of myoglobin remains unchanged while tertiary structure is lost significantly. 1-anilinonaphthalene-8-sulfonate binding experiments showed that myoglobin in the presence of various concentrations of ficoll 70, has newly exposed hydrophobic surfaces. Dynamic light scattering measurements show that there is almost 1.5 times increase in the hydrodynamic volume of myoglobin in the crowded environment. These structural characteristics of myoglobin in the presence of 300mg/ml ficoll 70 resemble those of molten globule state. Isothermal titration calorimetric (ITC) measurements show that ficoll 70 binds to myoglobin, whereas it shows no interaction with apo form of the protein. ITC results indicate that the reason behind this unique behavior of ficoll 70 towards myoglobin may be interaction of ficoll 70 with the heme group of myoglobin, which was further confirmed by the docking studies. We hypothesize that the soft interactions between heme and ficoll 70 leads to the formation of molten globule in myoglobin.

Molecular imprinting coupled with electrochemical analysis for plasma samples classification in acute myocardial infarction diagnostic.

Electroanalysis of myoglobin (Mb) in 10 plasma samples of healthy donors (HDs) and 14 plasma samples of patients with acute myocardial infarction (AMI) was carried out with screen-printed electrodes modified first with multi-walled carbon nanotubes (MWCNT) and then with a molecularly imprinted polymer film (MIP), viz., myoglobin-imprinted electropolymerized poly(o-phenylenediamine). The differential pulse voltammetry (DPV) parameters, such as a maximum amplitude of reduction peak current (A, nA), a reduction peak area (S, nA × V), and a peak potential (P, V), were measured for the MWCNT/MIP-sensors after their incubation with non-diluted plasma. The relevance of the multi-parameter electrochemical data for accurate discrimination between HDs and patients with AMI was assessed on the basis of electrochemical threshold values (this requires the reference standard method (RAMP® immunoassay)) or alternatively on the basis of the computational cluster assay (this does not require any reference standard method). The multi-parameter electrochemical analysis of biosamples combined with computational cluster assay was found to provide better accuracy in classification of plasma samples to the groups of HDs or AMI patients.

A method for extracting carboxy-myoglobin from beef.

The present study was conducted to devise a method for the effective extraction of carboxy-myoglobin (COMb) from beef without carbon monoxide dissociation. The ratio of COMb to myoglobin was computed at absorptions of wavelengths 541 and 551 nm, which characterize COMb and the isosbestic point between COMb and deoxy-myoglobin, respectively. The COMb extraction rate was found to vary with temperature, pH and oxygen conditions. The decrease observed in this rate was inversely proportional to the rise in extraction temperature. The COMb extraction rate was also affected by pH, and the stability of COMb in the extract solution was the highest at pH 8.0-9.0. Moreover, the presence of oxygen was found to disturb COMb extraction. According to these results, nearly all COMb could be extracted from carbon-monoxide-treated beef under stirring conditions in pH 8.5 deoxidized buffer, at 1°C, and under N2 flow with the improved extraction method in this study (98.1 ± 2.7%). The decrement of COMb in the extract was accelerated by light, and the COMb was stable for 20 min in the dark, at 1°C. The extraction conditions for COMb described above should allow the accurate evaluation of COMb in meat tissue.

Dynamic evaluation of a trilobal capillary-channeled polymer fiber shape for reversed phase protein separations and comparison to the eight-channeled form.

A new, trilobal-shaped capillary-channeled polymer fiber is under development to address the issues of poor A-term performance of the previous eight-channeled form. The trilobal geometry should provide better packing homogeneity due to the fewer potential orientations of the symmetric fiber geometry. Comparisons of separation efficiency and peak shape were made between the two fiber shapes through several dynamic parameters. Column hydrodynamics were investigated with two marker compounds, uracil and bovine serum albumin, with van Deemter plots of those two compounds revealing differences in the packing qualities between the different fiber shapes. Parametric fitting to the van Deemter, Knox, and Giddings equations provides insights into the column physical structures. Separation quality for both shapes was evaluated across differences in fiber packing density, gradient rate, and mobile phase linear velocity for the reversed phase separation of a four protein mixture, containing ribonuclease A, cytochrome c, lysozyme, and myoglobin. The results of this study lay the ground work for future efforts in the use of trilobal fibers for the separation of biomacromolecules.

Zinc oxide nanorods functionalized paper for protein preconcentration in biodiagnostics.

Distinguishing a specific biomarker from a biofluid sample containing a large variety of proteins often requires the selective preconcentration of that particular biomarker to a detectable level for analysis. Low-cost, paper-based device is an emerging opportunity in diagnostics. In the present study, we report a novel Zinc oxide nanorods functionalized paper platform for the preconcentration of Myoglobin, a cardiac biomarker. Zinc oxide nanorods were grown on a Whatman filter paper no. 1 via the standard hydrothermal route. The growth of Zinc oxide nanorods on paper was confirmed by a combination of techniques consisting of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS,) scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDX) analysis. The Zinc oxide nanorods modified Whatman filter paper (ZnO-NRs/WFP) was further tested for use as a protein preconcentrator. Paper-based ELISA was performed for determination of pre-concentration of cardiac marker protein Myoglobin using the new ZnO-NRs/WFP platform. The ZnO-NRs/WFP could efficiently capture the biomarker even from a very dilute solution (Myoglobin < 50 nM). Our ELISA results show a threefold enhancement in protein capture with ZnO-NRs/WFP compared to unmodified Whatman filter paper, allowing accurate protein analysis and showing the diagnostic concept.

Using an FPLC to promote active learning of the principles of protein structure and purification.

The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory exercise uses size exclusion chromatography (SEC) and ion exchange (IEX) chromatography to separate a mixture of four different proteins. Students use an SEC chromatogram and corresponding SDS-PAGE gel to understand how protein conformations change under different conditions (i.e. native and non-native). Students explore strategies to separate co-eluting proteins by IEX chromatography. Using either cation or anion exchange, one protein is bound to the column while the other is collected in the flow-through. In this exercise, undergraduate students gain hands-on experience with experimental design, buffer and sample preparation, and implementation of instrumentation that is commonly used by experienced researchers while learning and applying the fundamental concepts of protein structure, protein purification, and SDS-PAGE. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):60-68, 2017.

Proof of concept of a "greener" protein purification/enrichment method based on carboxylate-terminated carbosilane dendrimer-protein interactions.

Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample. Graphical Abstract Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional "no-clean" method based on acetone precipitation and the proposed "greener" method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio.