Sodium Dodecyl Sulfate Capillary Gel Research Articles

In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 μg/mL propidium iodide in the separation medium (LOD 2 μg/mL, 1.35 × 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 μg/mL, but using the local maximum at 100 μg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.

The Effect of Sample Glucose Content on PNGase F-Mediated N-Glycan Release Analyzed by Capillary Electrophoresis

Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure-function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly impacting the efficacy, immunogenicity, clearance rate, stability, solubility, pharmacokinetics and mode of action of the product. In most instances, these linked N-glycans are analyzed in their unconjugated form after endoglycosidase-mediated release, e.g., PNGase F-mediated liberation. In this paper, first, N-glycan release kinetics were evaluated using our previously reported in-house produced 6His-PNGase F enzyme. The resulting deglycosylation products were quantified by sodium dodecyl sulfate capillary gel electrophoresis to determine the optimal digestion time. Next, the effect of sample glucose content was investigated as a potential endoglycosidase activity modifier. A comparative Michaelis-Menten kinetics study was performed between the 6His-PNGase F and a frequently employed commercial PNGase F product with and without the presence of glucose in the digestion reaction mixture. It was found that 1 mg/mL glucose in the sample activated the 6His-PNGase F enzyme, while did not affect the release efficiency of the commercial PNGase F. Capillary isoelectric focusing revealed subtle charge heterogeneity differences between the two endoglycosidases, manifested by the lack of extra acidic charge variants in the cIEF trace of the 6His-PNGase F enzyme, which might have possibly influenced the glucose-mediated enzyme activity differences.

Electromigration Dispersion in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Proteins

The electromigration dispersion of the light- and heavy-chain subunit peaks of the therapeutic monoclonal antibody omalizumab was investigated in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran sieving matrices. Increasing boric acid content (340-640 mM) caused electromigration dispersion shifts for both low (2%)- and high (10%)-dextran-concentration gels in all gel-buffer compositions. In case of the heavy-chain fragment, elevated borate concentrations resulted in decreasing tailing and increasing fronting with the use of higher- and lower-dextran-concentration gels, respectively. The light-chain fragment, on the other hand, exhibited increased fronting with increasing borate concentration for both dextran concentrations examined in this study. Increase of the glycerol ingredient level in the gel-buffer system caused the same effect as the increasing borate concentration in both dextran concentrations. The detected electromigration dispersion was considered as the result of the formation of monomeric and dimeric glycerol-borate complexes as co-ionic constituents, migrating slower than that of the unconjugated tetrahydroxyborate. In addition, complexation of the tetrahydroxyborate anion with the glucose building blocks of the dextran polymer decreased its mobility to practically zero, contributing to further decrease in the resultant effective mobility of the co-ionic species. We suggest that the observed fronting and/or tailing peak shapes of the monoclonal antibody fragments in SDS-CGE at increasing boric acid concentrations can be considered as the result of multiple effects including changes in pH, sieving matrix pore size, viscosity, and the mobility variation of the co-ionic borate adducts with the gel-buffer ingredients. While electromigration dispersion-mediated band broadening, in general, can be minimized via matching the effective mobility of the co-ionic species to the analyte molecules of interest, in case of borate cross-linked dextran gels, optimization of the boric acid concentration required special consideration of its gel cross-linking function. For the light- and heavy-chain fragments of the IgG analyte, best peak shapes were attained with the use of 10% dextran/340 mM boric acid and 10% dextran/640 mM boric acid-containing gel-buffer systems, respectively. Based on this observation, here we introduce the concept of borate-gradient-mediated transient mobility matching in SDS-CGE of proteins. This novel approach resulted in close to optimal peak shapes for the distantly migrating IgG subunits within a single run, as well as unraveled the long-sought possible solution to perform capillary pore-size-gradient gel electrophoresis.

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels.

Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS-protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS-protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran-borate-glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (<0.96% relative standard deviation (RSD)). The widely used 10% dextran/4% borate gel represents a good screening option, which can then be followed by a modified composition, optimized for a specific separation as necessary.

Effect of the Monomer Cross-Linker Ratio on the Separation Selectivity of Monoclonal Antibody Subunits in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

With the increasing interest in the biopharmaceutical industry toward novel and innovative protein therapeutics, improved separation techniques are important, especially for the analysis of highly glycosylated candidates. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran is one of the most frequently used methods to analyze biotherapeutic proteins in process control as well as in release and stability testing. In this work, the effect of the monomer (dextran) and cross-linker (borate) ratio was studied in SDS-CGE analysis of a therapeutic monoclonal antibody test item in its reduced and intact forms. A retention model was developed for better understanding of the separation selectivity between the non-glycosylated and glycosylated heavy chain fragments, exploiting the interaction between the dextran-borate adducts and the glycan moiety of the therapeutic antibody. The monomer cross-linker ratio played a significant role in the overall analysis times and affected the separation selectivity between the non-glycosylated and regular (glycosylated) heavy chain fragments; however, it had no effect on the separation of the regular and non-glycosylated intact forms of the monoclonal antibody. Introduction of three-dimensional selectivity plots offered an easy separation optimization option for the separation problem in hand.

The Effect of Temperature in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Protein Therapeutics.

The temperature-dependent migration of molecular weight protein size standards and several biotherapeutic proteins were studied in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) in the interval from 15 to 60 °C using borate cross-linked dextran sieving matrix. Arrhenius plots were generated to calculate the respective activation energy values for the various solute molecules. SDS-CGE analysis of the biotherapeutic protein test mixture revealed no correlation between the activation energy requirement of the different species and their molecular weights, emphasizing the importance of separation temperature optimization to obtain high resolution between the solute molecules of interest. In contrast, the molecular weight protein size ladder ranging from 10 to 225 kDa, built from the same polypeptide blocks with no post-translational and other modifications, showed predictable activation energy requirement. The electrophoretic mobility of the SDS-protein complexes was found to be the function of the reciprocal sixth root of the molecular weight (Mw-1/6), implying cylindrical conformation.

Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins.

Size-based protein analysis utilizing only 25 ng of total proteins has been realized by sodium dodecyl sulfate capillary gel electrophoresis (SDS CGE) with head-column field-amplified sample stacking as an online sample preconcentration technique. This method has been used as a replacement of SDS-PAGE for purity analysis of adeno-associated virus (AAV) therapeutic products of different serotypes and transgenes. A limit of detection of 0.2 ng/mL (3.3 pM) capsid proteins was achieved with convenient UV absorbance detection at 214 nm, equivalent to 20 pg of protein (330 attomole) loaded in the autosampler vial. For purity analysis, only 25 ng of total AAV capsid proteins (4.3 femtomole virus particles) were loaded to the autosampler vial. The sensitivity is comparable to silver-stained SDS-PAGE. The RSD of purity measurement was 0.0-0.8%, comparable to conventional SDS CGE utilizing 0.1-0.5 mg proteins. The new method provided 3 orders of magnitude sensitivity enhancement as compared to conventional SDS CGE. It shares all the advantages of conventional SDS CGE (labor-saving, easy automation, and convenient quantitation) and also the high sensitivity of silver stained SDS-PAGE. The sample stacking SDS CGE technique can be adopted for size-based analysis of other types of proteins. It is especially useful when protein quantity or concentration is not sufficient for regular SDS CGE or SDS-PAGE assay.

Abstract 14609: Serum Protein Fingerprinting: A New Approach to the Clinical Screening of Coronary Artery Disease

Introduction: A major goal of the 21st century is to mitigate disease and healthcare costs. Coronary artery disease (CAD) kills 370,000 Americans and costs the United States $109 billion each year. More accurate screening tools are needed to ameliorate CAD related morbidity and mortality. A new approach to the direct analysis of blood serum led to the development of a prospective clinical screening tool for CAD involving sodium dodecyl sulfate (SDS) capillary gel electrophoresis (CGE). Hypothesis: The novel method generates serum protein profiles which can be used to screen individuals for heart disease by distinguishing profile features of macromolecules involved in CAD. Methods: Serum profiles were generated by SDS-CGE for each study participant (n=48), 30 with CAD and 18 ‘NonCAD’ as identified by histories of CAD or unremarkable coronary angiographies. Univariate and multivariate analyses of the profile features were conducted to identify biomarkers and statistically classify participants as CAD or NonCAD. To compare the accuracy of the method with that of an established clinical tool, participants’ 10 Year Framingham Risk Scores (FRS) were calculated. Individuals with FRS scores less than 10% (low) risk were classified as NonCAD and those with greater than 10% (moderate to severe) risk were classified as CAD. Results: A “serum protein fingerprint” profile is shown. Relative standard deviations of 2% for MW and 20% for absorbance indicate method repeatability. In the classification of participants as CAD or NonCAD, the respective accuracies of the multivariate canonical and linear discriminant analyses were 90% (p-value of 7E-5) and 94%. The classification accuracy of FRS was less than 42%. Two features of the serum profiles (#’s 3 and 7 (with Prob&gt;F values of 0.01 and 4E-5 respectively)) are potentially novel markers for CAD. Conclusions: The proposed serum protein analysis method correlated proteins with CAD and was more accurate than the FRS method in this study.

Protein concentration and bentonite requirement in Marlborough Sauvignon Blanc wines

Background and Aims Protein instability has long been a technical issue in white wine production. The aims of this study were: to examine the variability of wine proteins from a single cultivar within a defined wine region over two vintages; and to investigate the correlation of the content of the total and individual proteins and protein haze with the bentonite dosage required for heat stability. Methods and Results Proteins in Sauvignon Blanc juices and wines from five selected Marlborough, New Zealand, vineyards with two pruning/training regimes (two- and four-cane vertical shoot positioned) were studied over two consecutive vintages. Proteins were quantified and characterised by Coomassie Brilliant Blue assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis and sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE). The bentonite requirement for wine protein stability was determined by bentonite fining coupled with a hot/cold test. One vineyard consistently showed the lowest protein concentration and bentonite requirement regardless of pruning treatment and vintage, whereas others varied with pruning treatment and/or vineyard site and/or vintage. Two prevalent juice protein peaks at 22 and 28 kDa in SDS-CGE corresponded to two main wine protein peaks at 22 and 26 kDa, respectively. The 26 kDa fraction was reduced and became heterogeneous after fermentation, while the 22 kDa fraction remained unaffected. There was a good correlation between bentonite requirement and the 26 kDa fraction (R2 = 0.78). Conclusion The depletion of 26 kDa fraction in wine determined by SDS-CGE was a good indicator for protein stability by bentonite fining. Significance of the Study These results suggest that the method of protein quantification may have a strong influence on the utility of protein concentration measurement to predict the bentonite requirement of wine.

Capillary electrophoretic separation of high-molecular-weight poly(ethylene glycol)-modified proteins

This study was designed to demonstrate the utility of capillary electrophoresis (CE) for separating high-molecular-weight poly(ethylene glycol) (PEG)-conjugated proteins. As a CE method, sodium dodecyl sulfate–capillary gel electrophoresis (SDS–CGE) was applied to analyze interferon alpha (IFN) modified with branched and trimer-structured PEG molecules. Five mono-PEG-IFN conjugates prepared with two branched PEGs (MW 20 and 40 kDa) and three trimer-structured PEGs (MW 23.5, 43.5, and 47 kDa) were purified by cation-exchange chromatography and their masses were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The SDS–CGE method showed high separation capacity by differentiating PEG-IFN conjugates with small differences in molecular size, such as PEG 40K-, PEG 43.5K-, and PEG 47K-IFNs, and it was useful for checking the purity of each mono-PEG-IFN. This study shows that SDS–CGE can well be utilized in the development and quality control of PEGylated proteins prepared with various types of PEG.

Replaceable Cross-Linked Polyacrylamide for High Performance Separation of Proteins

An alternative sieving matrix, replaceable cross-linked polyacrylamide (rCPA), was developed for sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) separation of proteins. This rCPA could be conveniently pressurized into separation capillaries under a pressure of 80 psi. SDS-CGE separations using this matrix generated high resolutions for a wide range (approximately 4 kD to approximately 300 kD) of proteins. When compared to the most frequently used sieving matrixes, the rCPA permitted the highest resolutions with comparable or increased separation speed for protein separations.

High-throughput protein analysis by multiplexed sodium dodecyl sulfate capillary gel electrophoresis with UV absorption detection

High-throughput protein analysis by multiplexed sodium dodecyl sulfate capillary gel electrophoresis with UV absorption detection

High-throughput protein analysis by multiplexed sodium dodecyl sulfate capillary gel electrophoresis with UV absorption detection

We have developed a novel, high-throughput approach for molecular mass determination of proteins from 14 200 to 116 000 based upon multiplexed, absorbance-based capillary electrophoresis. Via capillary multiplexing, 96 samples were analyzed simultaneously within 30 min. Detection with ultraviolet light obviates the need for protein staining or derivatization. The detection limit of the system was estimated at 5 μg/ml bovine serum albumin (BSA) when sampled from 12.5 mM Tris–HCl. The linear dynamic range was over two orders of magnitude from 5 μg/ml to 1000 μg/ml for BSA. Better than 5% sizing accuracy for protein molecular mass determination and excellent run-to-run and day-to-day reproducibility was obtainable with the described method.

Capillary electrophoresis to characterize ricin and its subunits with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been employed as highly efficient methods to characterize ricin, its subunits, and the chemically deglycosylated forms. As a CE method, sodium dodecyl sulfate–capillary gel electrophoresis (SDS–CGE) was used because of its merit over the conventional slab gel techniques. SDS–CGE showed higher resolution capability over other analytical tools in the analysis of the ricin mixture as well as in each of its purified forms. The high resolution was considered to be a result of the presence of carbohydrates on ricin subunits, and this property was useful for identifying the native ricin or its A chain from their chemically deglycosylated forms. However, this method exhibited an overestimation of the molecular mass due to the carbohydrate moieties on ricin subunits, and the inaccuracies were observed to be dependent on the carbohydrate content of the subunits. The exact molecular masses were measured by MALDI-TOF MS, and the results were almost consistent with the expected values. This study clearly illustrates the usefulness and necessity of complementary use of two powerful analytical techniques to characterize ricin and its subunits in a various research fields such as poisoning and immunotoxin research.

Studies of Molecular Association of Glutenins and Maltodextrin in Solution by Capillary Electrophoresis

Molecular associations of wheat glutenins and carbohydrates are important in bread systems and believed to contribute to differences of a number of dough and baking quality characteristics. In this investigation, the study of molecular association of glutenins with carbohydrates in solution was performed by capillary zone electrophoresis (CZE). The carbohydrate used was a maltodextrin of average molecular weight 2000. To follow the molecular association of major glutenin subunits with the maltodextrin of interest, the extracted proteins from hard red winter wheat (cv. Scout 66) were fractionated first by reversed‐phase chromatography (RPC). Four major RPC fractions were collected, each of which still exhibited more than one glutenin component when reanalyzed by CZE. These RPC fractions were then incubated with the maltodextrin of interest, and the pre‐equilibrated glutenin‐maltodextrin complexes thus obtained were analyzed by CZE. Since maltodextrin is a neutral polymer, its binding to the glutenins results in decreasing the charge‐to‐mass ratio of the glutenins, and consequently, the effective electrophoretic mobility of protein–maltodextrin complex decreases with increasing maltodextrin concentration in the incubation solution. The evaluation of glutenin–maltodextrin interactions was only qualitative and the calculation of binding constants was hampered by various factors including the heterogeneity of the glutenin RPC fractions. The components of each RPC fraction yielded mobility lines (i.e., plots of electrophoretic mobility vs. percentage of maltodextrin in the glutenin sample) that paralleled each other indicating that these components have the same binding energetics magnitude with maltodextrin. This observation was substantiated by the sodium dodecyl sulfate capillary gel electrophoresis (SDS‐CGE) analysis, which showed that the various components of each RPC fraction are probably formed from one kind or one group of glutenin subunit with very similar properties. In addition, the mobility curves did not exhibit the usual rectangular hyperbolic forms of 1:1 binding isotherms.

Determination of biotin on a protein by quantitative sodium dodecyl sulfate–capillary gel electrophoresis of monomeric avidin

Sodium dodecyl sulfate–capillary gel electrophoresis (SDS–CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS–CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17 000. In the presence of a biotin–protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin and the initial amount is the portion of monomeric avidin bound to the conjugate. Accordingly, the number of biotin on the protein can be calculated. The assay is linearly responsive to increasing biotin loading in a biotinylation reaction of a protein. Accuracy of the assay is also demonstrated by good sample dilution recovery. Excellent quantitative reproducibility <2% (relative standard deviation) is obtained for both intra- and inter-day measurements. Main advantages of the method include the use of monomeric avidin that minimizes steric hindrance to capture biotin on a protein and assay automation on a capillary electrophoresis apparatus.

Determination of Vegetal Proteins in Milk Powder by Sodium Dodecyl Sulfate–Capillary Gel Electrophoresis: Interlaboratory Study

An interlaboratory study, with the participation of 8 laboratories, was conducted to evaluate a sodium dodecyl sulfate-capillary gel electrophoresis method for determination of adulteration of milk powder with soy and pea proteins. Calibration standards (0-8%, w/w, soy and pea protein in total protein) and adulterated skim milk powders (0-5%, w/w, soy and pea proteins in total protein) were produced. Vegetal proteins were determined after removal of milk proteins by pretreatment of the samples with tetraborate-EDTA buffer, pH 8.3. Repeatability standard deviations ranged from 9 to 15% and reproducibility standard deviations ranged from 25 to 30% in the samples containing 5% vegetal protein in total protein.

Characterization of the H- and L-Subunit Ratios of Ferritins by Sodium Dodecyl Sulfate–Capillary Gel Electrophoresis

Sodium dodecyl sulfate–capillary gel electrophoresis (SDS–CGE) was used to characterize the H- and L-subunit ratios of several mammalian ferritins and one bacterioferritin. Traditionally, SDS–PAGE has been used to characterize the H- and L-subunit ratios in ferritin; however, this technique is relatively slow and requires staining, destaining, and scanning before the data can be processed. In addition, the H- and L-subunits of ferritin are fairly close in molecular weight (∼21,000 and ∼20,000, respectively) and are often difficult to resolve in SDS–PAGE slab gels. In contrast, SDS–CGE requires no staining or destaining procedures and the peak quantitation is superior to SDS–PAGE. SDS–CGE is effective in quickly resolving the H- and L-subunits of ferritins from horse spleen, human liver, recombinant human H and L homopolymers, and mixtures of the two- and the single-subunit of a bacterioferritin from Escherichia coli. The technique has also proven useful in assaying the quality of the protein sample from both commercial and recombinant sources. Significant amounts of low-molecular-weight degradation products were detected in all commercial sources of horse spleen ferritin. Most commercial horse spleen ferritins lacked intact H-subunits under denaturing conditions.

Rational approach to quantitative sodium dodecyl sulfate capillary gel electrophoresis of monoclonal antibodies

Sodium dodecyl sulfate capillary gel electrophoresis has been used to separate and quantify murine monoclonal antibodies. The method uses a murine IgG, whose subclass differs from the analyte antibody, as an internal reference. The internal reference is chosen based on knowing that mouse IgG 1 can be separated from mouse IgG 2a or IgG 2b. Good intra- and inter-day reproducibility [relative standard deviation (RSD)<2%] of peak-area ratio has been obtained. A calibration curve also demonstrates high linearity ( R 2=0.9999) of response for the analyte. The described method is highly suitable for accurate determination of the antibody concentration even if a capillary electrophoresis apparatus is unable to provide good injection reproducibility.

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.