Sodium Dodecyl Sulfate Electrophoresis Research Articles

Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain

Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZ? plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture.

Physicochemical Characterization of Altebrel™, a Proposed Etanercept Biosimilar.

Background: Etanercept is prescribed for the rapid and effective treatment of chronic immune-mediated inflammatory disorders. Due to the expiration of etanercept patents and worldwide demand for comparable and more affordable substitutes, several biosimilars of etanercept have been approved in different countries and new ones are in the process of approval. Objectives: In the present study, Altebrel™ as an etanercept proposed biosimilar was investigated in a side by side comparison using various orthogonal analytical methods. Materials and Methods: Three batches of the Altebrel™ and Enbrel® samples were used for the study. Several physicochemical properties of samples were compared according to international guidelines, incliding; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion high performance liquid chromatography (SE-HPLC), hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and its biological activity was evaluated using surface plasmon resonance affinity analysis and tumor necrosis factor alpha (TNFα) neutralization biological assay. Amino acid analysis was applied to check the primary sequence and far-UV circular dichroism (CD) spectroscopy investigated the secondary structure. Results: The obtained results indicated a high degree of similarity between Altebrel™ and Enbrel®. Results of SDS-PAGE, CE-SDS, HIC-HPLC and SE-HPLC implied a comparable pattern of size variants for all samples. Based on the data achieved via in vitro bioactivity assays and SPR analysis, the functional property of Altebrel™ was proved comparable to that of the reference product. Moreover, amino acid analysis indicated similar primary structure and circular dichroism study implied a similar secondary structure for Altebrel™ and Enbrel®. Conclusion: Overall, our data provide analytical evidence for structural and in vitro functional similarity between Altebrel™ and Enbrel®.

Comparison of glycoprotein separation reveals greater impact of carbohydrates and disulfides on electrophoretic mobility for CE-SDS versus SDS-PAGE

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) has emerged as an indispensable tool in biopharmaceutical analysis to supersede the conventional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Despite the comparable migration behaviors of native proteins on the two platforms, the correlation has not been evaluated systematically for biotherapeutic proteins, which are often confounded by the presence of glycans and disulfides. Here we studied various mammalian glycoproteins using the two techniques under both reduced and non-reduced conditions. The results revealed substantial reduction in electrophoretic mobility under the capillary mode. Moreover, the migration order was found to be reversed between the reduced and nonreduced runs compared to SDS-PAGE. Such effects appeared to be independent of the content of sialylation. Our work unveiled complex interplays between the gel matrix, proteins and glycans which provide important guidance to biopharmaceutical analysis.

Characterization of ADCs by Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation technique that resolves ions based on their electrophoretic mobility in the presence of an applied voltage. It has been broadly applied for characterizing biotherapeutics including ADCs. In this chapter, step-by-step procedures for characterizing ADCs using CE will be described with focus placed on reduced and non-reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for purity determination and imaged capillary isoelectric focusing (iCIEF) for charge heterogeneity analysis.

Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation.

Positive identification of capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) electropherogram peaks provides information to understand protein molecular characteristics at the structural level. It is critical in the design of a robust assay that can accurately resolve, differentiate, and quantify all therapeutic protein components including fragmented species, which are considered as product related impurities. However, direct identification of the impurity peaks observed in CE-SDS is a challenging and oftentimes an ambiguous task. This paper proposed a systematic workflow for characterizing CE-SDS fragmentation peaks. Forced degradation of monoclonal antibody (mAb) by multiple stress methods was utilized to induce fragmentation and species enrichment. The characteristics, such as size and the clipped region of sequence, were then evaluated based on multiple enzymatic treatment and particle reduction. The identified fragments were further confirmed using tryptic digestion and liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Common fragment sizes and clipping locations are identified after evaluating multiple IgG molecules. The methodology and procedure described in this article are readily deployable and will provide necessary information for method, process, and product characterizations. Graphical abstract.

Glutathione S-Transferase: Purification and Characterization of from Cherry Laurel (Prunus laurocerasus L.) and the Investigation In Vitro Effects of Some Metal Ions and Organic Compounds on Enzyme Activity

In this study, the cherry laurel flesh fruit was purified to obtain glutathione S-transferase, a well-known antioxidant enzyme. Enzyme purification was performed using two separate processes: gel filtration and affinity chromatography. The sodium dodecyl sulfate electrophoresis method was used for determining enzyme purity. The optimum pH, optimum temperature, optimum ionic strength, stable pH, and KM and Vmax values for glutathione and 1-chloro-2,4-dinitrobenzene were obtained for the enzyme as follows: 7.0 in the K-phosphate buffer, 30 °C, 0.125 M, 6.5 in the K-phosphate buffer, 0.344 mM, 0.89 mM, and 0.098 EU/ml, 0.214 EU/ml. Lineweaver-Burk graphs were used to examine the in vitro enzyme activity in order to determine the inhibitory effects of some metal ions such as Cd2+, Ni2+, Cu2+, and Mg2+ and organic compounds such as sodium dodecyl sulfate (SDS), ascorbic acid (vitamin C), benzoic acid, and ethylenediaminetetraacetic acid (EDTA). The IC50 and Ki values for each of the metal ions and organic molecules were calculated. According to the results, Ca2+ metal ion and sodium dodecyl sulfate compounds were found to be the best inhibitor with a Ki value of 0.06 ± 0.04 and 0.20 ± 0.27, respectively.

Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening

Protein formulation stability is difficult to predict a priori and generally involves long-term stability studies. It is of interest to develop an analytical method that can predict stability trends reliably. Here, pulse proteolysis was evaluated as an analytical tool to predict solution-state stability in different formulations. Four proteins formulated in different buffer and excipient compositions were subjected to urea-induced unfolding and brief enzymatic digestion (“pulse” proteolysis), and relative resistance to proteolysis was measured by microfluidics-based capillary electrophoresis–sodium dodecyl sulfate. Biophysical properties of each formulation were measured using orthogonal biophysical techniques such as differential scanning fluorimetry, differential scanning calorimetry, dynamic light scattering, circular dichroism, and fluorescence spectroscopy. Protein stability in all formulations was monitored by size exclusion chromatography on storage at 5°C and 40°C. For all 4 proteins, formulations with greater proteolytic resistance also showed higher monomer content on thermal stability. In contrast, standard biophysical techniques showed reasonable-to-no correlation with size exclusion chromatography data. The data support the use of pulse proteolysis as an orthogonal, quantitative, and predictive tool to measure protein conformational stability and rank-order formulations.

Optimization of the cydex blue assay: A one-step colorimetric protein assay using cyclodextrins and compatible with detergents and reducers.

Sodium dodecyl sulfate electrophoresis (SDS) is a protein separation technique widely used, for example, prior to immunoblotting. Samples are usually prepared in a buffer containing both high concentrations of reducers and high concentrations of SDS. This conjunction renders the samples incompatible with common protein assays. By chelating the SDS, cyclodextrins make the use of simple, dye-based colorimetric assays possible. In this paper, we describe the optimization of the assay, focussing on the cyclodextrin/SDS ratio and the use of commercial assay reagents. The adaptation of the assay to a microplate format and using other detergent-containing conventional extraction buffers is also described.

Antibody-drug conjugate characterization by chromatographic and electrophoretic techniques.

Due to the inherent structure complexity and component heterogeneity of antibody drug conjugates (ADCs), separation technologies play a critical role in their characterization. In this review, we focus on chromatographic and electrophoretic approaches used to characterize ADCs with respect to drug-to-antibody ratio, drug distribution and conjugation sites, free small molecule drugs, charge variants, aggregates and fragments, etc. Chromatographic techniques including reversed-phase, ion exchange, size exclusion, hydrophobic interaction, two-dimensional liquid chromatography, and gas chromatography as well as capillary electrophoretic techniques including capillary electrophoresis sodium dodecyl sulfate, capillary zone electrophoresis and capillary isoelectric focusing are reviewed for their applications in the characterization of ADCs.

A Small-scale Model to Assess the Risk of Leachables from Single-use Bioprocess Containers through Protein Quality Characterization.

Leachables from single-use systems often exist in small quantities and are difficult to detect with existing analytical methods. The presence of relevant detrimental leachables from single-use bioprocess containers (BPCs) can be indirectly detected by studying the stability of monoclonal antibodies via changes by size exclusion chromatography, capillary electrophoresis sodium dodecyl sulfate, and visible/sub-visible particles using a small-scale stress model containing high surface area-to-volume ratio at elevated temperature alongside with an appropriate control (e.g., glass vials or stainless steel containers). These changes in protein quality attributes allowed the evaluation of potential risks associated with adopting single-use bioprocess containers for storage as well as bag quality and bag differences between earlier and later batches. These leachables appear to be generated during the bag sterilization process by gamma irradiation. Improvements in protein stability after storage in "preheated" bags indicated that these leachables may be thermally unstable or volatile. The effect of surfactant levels, storage temperatures, surface area-to-volume ratios, filtration, and buffer exchange on leachables and protein stability were also assessed.

Implementation of USP antibody standard for system suitability in capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for release and stability methods

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is widely used for purity analysis of monoclonal antibody therapeutics for release and stability to demonstrate product consistency and shelf life during the manufacturing and life cycle of the product. CE-SDS method development is focused on exploring the method capability to provide the information about the product purity and product related degradants (fragmentation, aggregation etc.). In order to establish the functionality of the instrumentation, software, and sample preparation; system suitability criteria need to be defined for analytical methods using a well characterized reference standard run under the same protocol and analysis as the test articles. Typically the reference standard is produced using a manufacturing process representative of the clinical material. The qualification, control, and maintenance of in-house reference standards are established through rigorous quality and regulatory guidelines. The U.S. Pharmacopeia (USP) has developed a monoclonal IgG System Suitability Reference Standard to be utilized for assessment of system suitability in CE-SDS methods.In this communication, we evaluate the system suitability acceptance criteria performance of the USP IgG standard using two methods, the recommended USP protocol provided in monograph <129> and a molecule specific Bristol-Myers Squibb (BMS) CE-SDS method. The results from USP IgG standard were compared with two in-house monoclonal antibody reference standards. The data suggest that the USP CE-SDS method may not be suitable for CE-SDS analysis for release and stability of monoclonal antibody therapeutics due to the high level of method induced partial reduction observed for all molecules tested. This high level of fragmentation observed utilizing the USP method will result in reporting lower purity levels, which will impact the overall quality assessment of the molecule. The system suitability criteria recommended by the USP method can be achieved by using the USP reference standard during the development and pre-validation stages. Furthermore, the USP reference standard does not offer significant advantages to existing SST criteria in the BMS method during release and stability testing, and therefore we propose use of the USP standard only during the optimization and pre-validation stages of method development.

Amyloid fibrillogenesis of lysozyme is suppressed by a food additive brilliant blue FCF

At least 30 different human proteins can fold abnormally to form the amyloid deposits that are associated with a number of degenerative diseases. The research presented here aimed at understanding the inhibitory potency of a food additive, brilliant blue FCF (BBF), on the amyloid fibril formation of lysozyme. Our results demonstrated that BBF was able to suppress the formation of lysozyme fibrils in a dose-dependent fashion. In addition, the structural features and conformational changes in the lysozyme samples upon the addition of BBF were further characterized using circular dichroism spectroscopy, nile red fluorescence spectroscopy, turbidity assay, and sodium dodecyl sulfate electrophoresis. Through molecular docking and molecular dynamics simulations, BBF’s mechanism of action in lysozyme fibrillogenesis inhibition was found to be initiated by binding with the aggregation-prone region of the lysozyme. We believe the results from this research may contribute to the development of effective therapeutics for amyloidoses.

Application of Microchip Electrophoresis Sodium Dodecyl Sulfate for the Evaluation of Change of Degradation Species of Therapeutic Antibodies in Stability Testing

We evaluated the performance of a commercial microchip electrophoresis instrument (LabChip(®) GXII) for the evaluation of change of degradation species of therapeutic antibodies in stability testing. This system requires a sample volume of only 5 μg, and indicates fine resolution of size variant species such as light chain, heavy chain, non-glycosylated heavy chain and various degradation species. Precision and accuracy were high; the intermediate precision of 18 determinations was only 2.1% or less as RSD and recoveries ranged from 97.8 to 103.0% for major species as heavy chain, light chain and intact molecule of a therapeutic antibody. The applicability of this method was demonstrated by applying the method for the analysis of heat-degraded products of three pharmaceutical antibodies. Though some fragment peaks commonly appeared and increased according to temperature regardless of the source of preparations, one of them indicated specific peaks implying the cleavage of the peptide chain of the heavy chain. We also compared the performance of the method with those using conventional capillary-based SDS electrophoresis. Although the absolute purity values expressed as peak area % were different for the two methods, probably due to the difference in the detection methods, similar quality profiles were obtained within 40 s by microchip-based SDS electrophoresis. In addition, the degradation manner of three marketed antibodies depending on temperature was almost the same for the two methods. At the first stage in the development of manufacturing antibody pharmaceuticals, various factors including cell selection, cell cultivation, and formulation development should be evaluated using limited sample amounts. The stability testing using microchip-based electrophoresis seems suitable for these purposes.

Characterization of a Novel Cross-Linked Lipase: Impact of Cross-Linking on Solubility and Release from Drug Product

Liprotamase is a novel non-porcine pancreatic enzyme replacement therapy containing purified biotechnology-derived lipase, protease, and amylase together with excipients in a capsule formulation. To preserve the structural integrity and biological activity of lipase (the primary drug substance) through exposure of the drug product to the low-pH gastric environment, the enzyme was processed through the use of cross-linked enzyme crystal (CLEC) technology, making the lipase-CLEC drug substance insoluble under acidic conditions but fully soluble at neutral pH and in alkaline environments. In this report we characterize the degree of cross-linking for lipase-CLEC and demonstrate its impact on lipase-CLEC solubility and release from the drug product under relevant physiological pH conditions. Cross-linked lipase-CLEC was characterized via size exclusion chromatography (SEC) and capillary electrophoresis sodium dodecyl sulfate polyacrylamide gel electrophoresis (CE-SDS-PAGE). A combination of methodologies was developed to understand the impact of cross-linking on drug product release. Dissolution evaluation using USP Apparatus 2 at pH 5.0 with an enzyme activity-based end point demonstrated solubility discrimination based on degree of cross-linking, while full release was demonstrated at pH 6.5. The dissolution of the drug product was also evaluated using a dual-stage test employing a USP Apparatus 4 flow-through system to mimic the changing pH environments experienced in the stomach and intestine to understand the impact of cross-linking on drug product performance. Use of USP Apparatus 4 to characterize the pH-dependent release of lipase-CLEC represents a novel approach compared to the Apparatus 1 test employing an acid-challenge stage outlined in the USP for delayed-release pancrelipase, and the advantages of this approach may prove useful for understanding the pH-dependence of release for other drug products. Collectively, these studies confirmed that degree of cross-linking is a critical parameter that may impact in vivo release of lipase-CLEC, and also provided a risk assessment tool for understanding the potential impact of under- and over-cross-linked drug substance.

The Detection of Complexed Proteins E6/P53 and E7/Prb In Relation to Carcinogenesis of the Uterine Cervix

Objective: The aim of our work was to identify by Western Blot technique (WB) the oncoproteins generated by E6 and E7 genes of high risk Human Papillomavirus (HR-HPV), resulting from integration of viral genomes into cervical cell DNA and their interactions with tumor suppressor human proteins p53 and pRb in premalignant or malignant cervical lesions. Methods: The study was performed on 2,500 women from Caserta Local Health Authority who underwent cervical cytology from June 2010 to September 2011. Informed consent of participants was obtained. The cell samples taken by brushing were stored in liquid based cytology (LBC) and in physiological solution for WB analysis. Cytology diagnoses was based on 2001 Bethesda classification. Proteomic research was performed according to size after extraction and SDS (Sodium Dodecyl Sulfate) electrophoresis. Proteins of interest were detected by primary monoclonal antibodies and WB analysis. Results: Cytology results of 2,500 women were positive for abnormalities of epithelial cells in 3.1%. Atypical squamous cells of undetermined significance (ASC-US) were 1.3%, Low Squamous Intraepithelial Lesion (L-SIL) 1.7% and High Squamous Intraepithelial Lesion (H-SIL) or worse 0.2%. The integration of E6 and E7 genes with their oncoproteins expression were found in 3.1% of ASC-US and in all cases of SIL or worse. On the other hand positive interactions of E6/p53 proteins and E7/pRb were found in 4.8% of L-SIL, in 75.0% of H-SIL or worse and in no ASC-US. Conclusions: Results show that the integration of viral genomes (E6 and E7) is present in all H-SIL or worse and in several L-SIL, but only the most advanced lesions, histologically confirmed, have shown the E6/p53 interaction and E7/pRb. Our protocol allows a selection of women with advanced lesions toward cancer to obtain appropriate management.

Characterization of somatic antigens of adult Toxocara canis by western blotting

Aim: The objective of this study was characterize the somatic soluble antigens of adult Toxocara canis (Tc-SA) by western blotting. Materials and Methods: T. canis worms were collected from the naturally infected pups after deworming. The somatic antigen was prepared as per standard procedure with slight modification. These antigens were separated using Sodium dodecyl sulphate-electrophoresis (SDS-PAGE). The specific reactivity of the Tc-SA proteins was checked against the serum of naturally infected dogs as well with the hyperimmune serum raised in the rabbit by western blotting. Results: On SDS-PAGE recovered proteins ranged in size from 44 to 300 kDa. The immuno-reactivity of the naturally infected dog sera with the Tc-SA antigens showed 12 prominent immunoreactive bands of distinct sizes at 28.61, 32.60, 38.10, 43.04, 49.99, 67.57, 73.22, 105.77, 144.74, 161.11, 177.84 and 196.31 kDa. The immuno-reactivity of the hyper immune serum raised in rabbits against Tc-SA antigens was observed with 10 prominent bands of distinct sizes at 17.11, 24.15, 34.83, 43.46, 52.47, 55.89, 67.57, 70, 74.60 and 105.6 kDa. Conclusions: Common antigens band were observed at 67 and 105 kDa. These antigens merit further evaluation as candidate for use in diagnosis of toxocarosis in humans and adult dogs.

Characterization of excretory-secretory antigens of adult Toxocara canis by western blotting

Toxocara canis is one of the most common helminth worm of dogs which continues to stimulate both public health concern alongside the higher scientific interest. It may cause visceral and ocular damage in humans especially in children. The identification of specific antigens of T. canis is important so as to develop better diagnostic techniques. Excretory-secretory (ES) antigens were prepared by culturing the adult T. canis worms in RPMI 1640 medium without serum supplementation followed by ammonium sulphate precipitation. These antigens were separated using sodium dodecyl sulphate-electrophoresis (SDS-PAGE). Recovered proteins ranged from 30 to 384kDa. The specific reactivity of the T. canis excretory-secretory (TC-ES) proteins was checked by western blotting. The immuno-reactivity of the naturally infected dog sera with the TC-ES antigens showed five bands at 43, 57,105, 139 and 175kDa. The immuno-reactivity of the hyper immune serum raised in rabbits against TC-ES antigens was observed with ten polypeptides of 21, 25, 30, 37, 45, 50, 57, 69, 77 and 105kDa. Common antigens band were observed at 57 and 105KDa. These antigens merit further evaluation as candidate for use in diagnosis of toxocariasis in humans and adult dogs.

Allelic diversity of high molecular weight glutenin subunits (HMW-GS) in Iranian Aegilops tauschii Coss. accessions by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

Variation of high molecular weight glutenin subunits (HMW-GS) in 28 Iranian Aegilops tauschii (2n = 2x = 14, DD) accessions studied by sodium dodecyl sulphate electrophoresis method (SDS-PAGE). The results showed high variation of HMW-GS in the accessions. The range of frequency in 14 HMW-GS combinations was 3.57–25 % in the accessions. AMOVA showed the molecular variance between the geographic areas was lower than within the geographic areas. According to Nei’s genetic diversity, the highest diversity levels were in Semnan, Golestan and Azarbayjan, on the other hand the lowest levels of diversity were found in Khorasan, Gilan and Mazandaran accessions. Hence, the Caspian Sea South East accessions also Azerbayjan in Iran have more diversity. AMOVA did not show variance between strangulata and tauschii but there was more genetic diversity in ssp. tauschii subspecies in comparison of ssp. strangulata according to Nei’s gene diversity and Shannon information index. It showed Iranian Ae. tauschii have a good potential for bread making quality improvement in bread wheat.

Characterization of glycation in an IgG1 by capillary electrophoresis sodium dodecyl sulfate and mass spectrometry

We report a case study of characterization of a non-enzymatically glycated IgG1 using reducing capillary electrophoresis sodium dodecyl sulfate (CE–SDS) and mass spectrometry (MS). Glycation was found to occur nonspecifically at multiple sites in both the light and heavy chains. The glycated light and heavy chains result in wider peaks eluting late in the reducing CE–SDS profile; in particular, the glycated light chain behaved as a shoulder peak detected by either ultraviolet (UV) or laser-induced fluorescence (LIF) signals. The glycated species can be enriched by boronate affinity chromatography. Analyzing the enriched samples by reversed phase high-performance liquid chromatography in line with time-of-flight MS (RP–HPLC–TOF/MS) revealed adducts of +162 and +324 Da to both the light and heavy chains, suggesting the presence of multiple glycation sites. Tryptic peptide mapping and tandem mass sequencing were used to identify two glycation sites on each of the light and heavy chains.

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography

The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C.3.1.1.7) enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium's pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A-Sepharose 4B and edrophonium-Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.