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Lab on a Chip Dec 2017Cell-matrix and cell-cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from...
Cell-matrix and cell-cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from the surrounding microenvironment alters intracellular signalling. Here, we demonstrate and characterise an integrated microfluidic device to culture single and clustered cells in tuneable microenvironments and then directly analyse the lysate of each cell in situ, thereby eliminating the need to detach cells prior to analysis. First, we utilise microcontact printing to pattern cells in confined geometries. We then utilise a microscale isoelectric focusing (IEF) module to separate, detect, and analyse lamin A/C from substrate-adhered cells seeded and cultured at varying (500, 2000, and 9000 cells per cm) densities. We report separation performance (minimum resolvable pI difference of 0.11) that is on par with capillary IEF and independent of cell density. Moreover, we map lamin A/C and β-tubulin protein expression to morphometric information (cell area, circumference, eccentricity, form factor, and cell area factor) of single cells and observe poor correlation with each of these parameters. By eliminating the need for cell detachment from substrates, we enhance detection of cell receptor proteins (CD44 and β-integrin) and dynamic phosphorylation events (pMLC) that are rendered undetectable or disrupted by enzymatic treatments. Finally, we optimise protein solubilisation and separation performance by tuning lysis and electrofocusing (EF) durations. We observe enhanced separation performance (decreased peak width) with longer EF durations by 25.1% and improved protein solubilisation with longer lysis durations. Overall, the combination of morphometric analyses of substrate-adhered cells, with minimised handling, will yield important insights into our understanding of adhesion-mediated signalling processes.
Topics: Biomarkers; Cell Adhesion; Cell Line; Flow Cytometry; Humans; Isoelectric Focusing; Microfluidic Analytical Techniques
PubMed: 29120467
DOI: 10.1039/c7lc01012e -
Analytical and Bioanalytical Chemistry May 2009Free-flow electrophoresis (FFE) is a technique that performs an electrophoretic separation on a continuous stream of analyte as it flows through a planar flow channel.... (Review)
Review
Free-flow electrophoresis (FFE) is a technique that performs an electrophoretic separation on a continuous stream of analyte as it flows through a planar flow channel. The electric field is applied perpendicularly to the flow to deflect analytes laterally according to their mobility as they flow through the separation channel. Miniaturization of FFE (microFFE) over the past 15 years has allowed analytical and preparative separation of small volume samples. Advances in chip design have improved separations by reducing interference from bubbles generated by electrolysis. Mechanisms of band broadening have been examined theoretically and experimentally to improve resolution in microFFE. Separations using various modes such as zone electrophoresis, isoelectric focusing, isotachophoresis, and field-step electrophoresis have been demonstrated.
Topics: Electrophoresis; Isoelectric Focusing; Sensitivity and Specificity
PubMed: 19290514
DOI: 10.1007/s00216-009-2656-5 -
Molecules (Basel, Switzerland) May 2023Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using... (Review)
Review
Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.
Topics: Reproducibility of Results; Erythropoietin; Body Fluids; Isoelectric Focusing; Blotting, Western; Antibodies; Electrophoresis, Polyacrylamide Gel; Substance Abuse Detection; Recombinant Proteins
PubMed: 37298922
DOI: 10.3390/molecules28114446 -
Se Pu = Chinese Journal of... Jul 2022This paper provides an annual review of capillary electrophoresis (CE) technology in 2021. A total of 291 research papers related to CE technology published in 2021 were... (Review)
Review
This paper provides an annual review of capillary electrophoresis (CE) technology in 2021. A total of 291 research papers related to CE technology published in 2021 were retrieved from the ISI Web of Science using the keywords, "capillary electrophoresis-mass spectrometry" "capillary isoelectric focusing" "micellar electrokinetic chromatography", or "capillary electrophoresis" (not "capillary electrochromatography" "microchip" and "capillary monolithic column"). In addition, nine research papers related to CE technology in Chinese journals were reviewed: and . This review focused on seven papers published in , , , , and with impact factors (IFs) greater than 10.0, as well as 42 papers reported in , , , and with IFs between 5.0 and 10.0. This review also provides a comprehensive overview of representative CE works in and with IFs<5.0, as well as important Chinese journals, and . According to the IF, this paper introduces the representative work of CE-related papers to allow readers to quickly understand the important research progress of CE technology in the past year.
Topics: Capillary Electrochromatography; Food; Isoelectric Focusing; Micelles; Technology
PubMed: 35791597
DOI: 10.3724/SP.J.1123.2022.03040 -
Biochemia Medica 2012Biochemical and biological properties of glycoconjugates are strongly determined by the specific structure of its glycan parts. Glycosylation, the covalent attachment of... (Review)
Review
Biochemical and biological properties of glycoconjugates are strongly determined by the specific structure of its glycan parts. Glycosylation, the covalent attachment of sugars to proteins and lipids, is very complex and highly-coordinated process involving > 250 gene products. Deficiency of glycosylation enzymes or transporters results in impaired glycosylation, and consequently pathological modulation of many physiological processes. Inborn defects of glycosylation enzymes, caused by the specific mutations, lead to the development of rare, but severe diseases - congenital disorders of glycosylation (CDGs). Up today, there are more than 45 known CDGs. Their clinical manifestations range from very mild to extremely severe (even lethal) and unfortunately, only three of them can be effectively treated nowadays. CDG symptoms highly vary, though some are common for several CDG types but also for other unrelated diseases, especially neurological ones, leaving the possibility that many CDGs cases are under- or misdiagnosed. Glycan analysis of serum transferrin (by isoelectric focusing or more sophisticated methods, such as HPLC (high-performance liquid chromatography) or MALDI (matrix-assisted laser desorption/ionization)) or serum N-glycans (by MS), enzyme activity assays and DNA sequence analysis are the most frequently used methods for CDG screening and identification, since no specific tests are available yet. In this review we summarize the current knowledge on the clinical, biochemical and genetic characteristic of distinct CDGs, as well as existing diagnostic and therapeutic procedures, aiming to contribute to the awareness on the existence of these rare diseases and encourage the efforts to elucidate its genetic background, improve diagnostics and develop new strategies for their treatment.
Topics: Biochemistry; Chromatography, High Pressure Liquid; Clinical Laboratory Techniques; Congenital Disorders of Glycosylation; Female; Glycosylation; Humans; Isoelectric Focusing; Male; Mass Spectrometry; Models, Genetic; Mutation; Polysaccharides; Pregnancy; Prenatal Diagnosis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 22838182
DOI: 10.11613/bm.2012.019 -
The Indian Journal of Medical Research Oct 2011Haemoglobin (Hb) abnormalities though quite frequent, are generally detected in populations during surveys and programmes run for prevention of Hb disorders. Several... (Review)
Review
Haemoglobin (Hb) abnormalities though quite frequent, are generally detected in populations during surveys and programmes run for prevention of Hb disorders. Several methods are now available for detection of Hb abnormalities. In this review, the following are discussed: (i) the methods used for characterization of haemoglobin disorders; (ii) the problems linked to diagnosis of thalassaemic trait; (iii) the strategy for detection of common Hb variants; and (iv) the difficulties in identification of rare variants. The differences between developing and industrialized countries for the strategies employed in the diagnosis of abnormal haemoglobins are considered. We mention the limits and pitfalls for each approach and the necessity to characterize the abnormalities using at least two different methods. The recommended strategy is to use a combination of cation-exchange high performance chromatography (CE-HPLC), capillary electrophoresis (CE) and when possible isoelectric focusing (IEF). Difficult cases may demand further investigations requiring specialized protein and/or molecular biology techniques.
Topics: Chromatography, High Pressure Liquid; Electrophoresis, Capillary; Erythrocytes; Genetic Variation; Hemoglobinopathies; Hemoglobins, Abnormal; Humans; Isoelectric Focusing; Phenotype; beta-Thalassemia
PubMed: 22089618
DOI: No ID Found -
Scientific Reports Nov 2023Ferritin is a ubiquitous intracellular iron storage protein that plays a crucial role in iron homeostasis. Animal tissue ferritins consist of multiple isoforms (or...
Ferritin is a ubiquitous intracellular iron storage protein that plays a crucial role in iron homeostasis. Animal tissue ferritins consist of multiple isoforms (or isoferritins) with different proportions of H and L subunits that contribute to their structural and compositional heterogeneity, and thus physiological functions. Using size exclusion and anion exchange chromatography, capillary isoelectric focusing (cIEF), and SDS-capillary gel electrophoresis (SDS-CGE), we reveal for the first time a significant variation in ferritin subunit composition and isoelectric points, in both recombinant and native ferritins extracted from animal organs. Our results indicate that subunits composition is the main determinant of the mean pI of recombinant ferritin heteropolymers, and that ferritin microheterogeneity is a common property of both natural and recombinant proteins and appears to be an intrinsic feature of the cellular machinery during ferritin expression, regulation, post-translational modifications, and post-subunits assembly. The functional significance and physiological implications of ferritin heterogeneity in terms of iron metabolism, response to oxidative stress, tissue-specific functions, and pathological processes are discussed.
Topics: Animals; Ferritins; Isoelectric Focusing; Electrophoresis, Polyacrylamide Gel; Iron; Isoelectric Point
PubMed: 37963965
DOI: 10.1038/s41598-023-46880-9 -
Nucleic Acids Research Jul 2021The isoelectric point is the pH at which a particular molecule is electrically neutral due to the equilibrium of positive and negative charges. In proteins and peptides,...
The isoelectric point is the pH at which a particular molecule is electrically neutral due to the equilibrium of positive and negative charges. In proteins and peptides, this depends on the dissociation constant (pKa) of charged groups of seven amino acids and NH+ and COO- groups at polypeptide termini. Information regarding isoelectric point and pKa is extensively used in two-dimensional gel electrophoresis (2D-PAGE), capillary isoelectric focusing (cIEF), crystallisation, and mass spectrometry. Therefore, there is a strong need for the in silico prediction of isoelectric point and pKa values. In this paper, I present Isoelectric Point Calculator 2.0 (IPC 2.0), a web server for the prediction of isoelectric points and pKa values using a mixture of deep learning and support vector regression models. The prediction accuracy (RMSD) of IPC 2.0 for proteins and peptides outperforms previous algorithms: 0.848 versus 0.868 and 0.222 versus 0.405, respectively. Moreover, the IPC 2.0 prediction of pKa using sequence information alone was better than the prediction from structure-based methods (0.576 versus 0.826) and a few folds faster. The IPC 2.0 webserver is freely available at www.ipc2-isoelectric-point.org.
Topics: Deep Learning; Isoelectric Point; Peptides; Proteins; Sequence Analysis, Protein; Software; Support Vector Machine
PubMed: 33905510
DOI: 10.1093/nar/gkab295 -
Journal of Pharmaceutical and... Jan 2023This review provides a comprehensive overview of methodological advances and applications of CE in the analysis and characterization of recombinant therapeutic and... (Review)
Review
This review provides a comprehensive overview of methodological advances and applications of CE in the analysis and characterization of recombinant therapeutic and diagnostic proteins over the past two decades. The first part of the review discusses various aspects of biotechnological protein production and the related effects on the final product. This covers upstream processes, e.g., selection and transfection of host cells, up-scaling of cell cultures and cultivation conditions, as well as downstream processing and a discussion of future trends in biotechnological manufacturing. This part is essential for relating biotechnological production to analytical challenges and requirements in order to provide a holistic insight. In this context, the influence of manufacturing steps on the quality of the final drug substance/product is discussed in terms of related post-translational modifications of the target molecule with a major focus on glycosylation pattern and conformational effects. Particular attention is given to host cell specific and non-human modifications affecting the efficacy and safety of recombinant products. Endowed with this propaedeutic knowledge, the major part of the review discusses the manifold contributions of different CE techniques to the development and optimization of the manufacturing process, to the evaluation and characterization of the final drug product and their role in quality control. Different CE techniques, such as CZE, capillary gel electrophoresis (CGE), (imaged) capillary isoelectric focusing ((i)CIEF), µChipCE, CE-Western blot, affinity CE (ACE), and CE-MS are discussed including a brief introduction in the respective separation and hyphenation principle as well as their applications in the analysis of different recombinant biologics together with recent strategies. The addressed analyte portfolio comprises a vast variety of recombinant proteins with molecular masses from 4.1 kDa up to 20.3 MDa (for recombinant virus-like particles), and a pI range from 2.0 to 11.2. Antibodies are not explicitly covered in the survey. The review is complemented by compiling validation aspects and proposed suitability tests in order to assure the feasibility of methods to industrial and pharmaceutical needs.
Topics: Mass Spectrometry; Isoelectric Focusing; Electrophoresis, Capillary; Recombinant Proteins; Biological Products
PubMed: 36279846
DOI: 10.1016/j.jpba.2022.115089 -
PloS One 2016We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements... (Comparative Study)
Comparative Study
Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting.
OBJECTIVES
We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups.
METHODS
We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing.
RESULTS
Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF.
CONCLUSIONS
Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.
Topics: Case-Control Studies; Demyelinating Diseases; Enzyme-Linked Immunosorbent Assay; Humans; Immunoblotting; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Isoelectric Focusing; Multiple Sclerosis; Nephelometry and Turbidimetry; Observer Variation; ROC Curve; Reproducibility of Results
PubMed: 27846293
DOI: 10.1371/journal.pone.0166556