Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Technique Research Articles

Investigation of genetic diversity in lentil genotypes obtain from different countries using SDS-PAGE methodology

Pulses are an important component of the agricultural production system, providing high-quality food to millions of people worldwide. Among pulses, lentil is an annual and self-pollinated crop known to be its high proteins, amino acids, dietary fibers and low fat in the world. In the current study, the characterization of 44 lentil genotypes was performed using SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) technique. The protein bands were scored according to a binary system as the present (1) or absent (0) that ranged 12-20 bands in lentil genotypes. The Jaccard’s coefficient of genetic dissimilarity (GD) was measured, and the mean GD was 0.216. Maximum genetic distance was found as 0.526 between Syria2 with Iraq3 and these genotypes might be recommended for future lentil breeding. AMOVA explored the presence of higher genetic variety within genotypes (83%) than among genotypes (17% variations). STRUCTURE algorithm separated lentil genotypes into two groups mainly on the basis of their geographic. The UPGMA clustering separated lentil genotypes into two main clusters A and B. The PCoA was also conducted to confirm the results of structure and UPGMA analyses. Findigs from the PCoA and structure analyses were in full agreement with those obtained by UPGMA. The results might be useful for researchers worldwide who are interested in lentil breeding.

Stability and Biosimilarity Assessment of Bevacizumab Monoclonal Antibody; Orthogonal Testing Protocol Coupled With Peptide Mapping-Principal Component Analysis.

Biologics are essential in cancer treatment because they stimulate the body's natural response to fight cancer, but they are expensive. Biosimilars are more affordable compared to patent biologicals, but it must be verified that they are as effective as their innovators. Characterization of biosimilars and assessment of interchangeability requires many data points for verification. The proposed study provides a quality assessment of two new bevacizumab (BVZ) biosimilars, produced by Amgen and Biocad, Inc., through the development and greenness assessment of an orthogonal testing protocol and purity indicating assay, including size-exclusion (SE-HPLC), reversed-phase (RP-HPLC), and cation exchange chromatography (CEX-HPLC) in addition to dynamic light scattering (DLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SE-HPLC method was performed and validated to screen the BVZ monomer and its aggregates and/or fragments. Peak purity and system suitability parameters were calculated. Results indicate that the orthogonal protocol is a useful tool for assessing monoclonal antibody stability. It is a key criterion for biosimilarity assessment. DLS and SDS-PAGE results were compared to each other to reveal close retention times and banding patterns between BVZ innovator and its biosimilars. These results showed that Avastin® and the investigated biosimilars have the same profile in terms of peak area of related compounds within the acceptance limit and apparent molecular weight, and the SDS-PAGE technique was found to be the most eco-friendly technique among others. The results obtained highlighted the importance of assessing similarities and differences in ensuring the biosimilarity and interchangeability of the studied products. BVZ is one of the essential monoclonal antibodies in the treatment of colorectal cancer (CRC). BVZ biosimilars were evaluated by developing an orthogonal testing protocol and a purity-indicating assay. The size-exclusion (SE)-HPLC method was applied and validated to monitor the BVZ monomer and its aggregates. The results demonstrated the importance of assessing the stability and biosimilarity of BVZ.

Enhancement of resilient modulus of cohesive soil using an enzymatic preparation

The current study aims to evaluate the dynamic response of stabilized cohesive soil using an enzymatic preparation in terms of resilient modulus. We ran a series of resilient modulus testing according to AASHTO T307 on three types of cohesive soil treated with an enzymatic preparation to investigate its potential on roads construction. The results show significant improvement in the resilient modulus values, estimated at 1.4 to 4.4 times that observed for the untreated soil. Because of the complexity in conducting the resilient modulus measurement, we did a regression analysis to produce reliable correlation formula to predict the resilient modulus for untreated and stabilised soil samples involving stress state. The resilient modulus values for the subgrade materials at the anticipated field stresses were determined using a universal model. The enzymatic preparation was applied in pavement of a sample road and evaluated using the plate load test. SEM analysis for soil samples shows improvement in the soil compaction via reduction of voids between soil particles. XRD analysis shows no major structural changes in the treated soils. The enzymatic preparation contains 43 mg/mL of proteins. We used the SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) technique to identify the main protein components; however, the presence of interfering materials (surfactants) hinders the separation.

Analysis of Whole Cell Protein Profiles by SDS-PAGE to Identify Indigenous Cellulose-producer Acetic Acid Bacteria

This study was carried out to analyze the suitability of the identification of four indigenous cellulose-producing acetic acid bacterial isolates (ANG29, KRE65, ANG32 and SAL53) based on the analysis of whole cellular protein profiles against identification based on phenotypic traits. Whole cellular protein profiles were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. The whole cellular protein profiles obtained from sample isolates, were compared with reference isolates for species identification. The results showed that based on visual observations can be determined as much as 12 bands of protein with a molecular weight of 19,099 KDa up to 132.182 KDa. Based on the analysis of protein bands were detected visually, fourth indigenous cellulose- producing acetic acid bacterial isolates in the study had a higher similarity profile to the reference strain Gluconacetobacter xylinus BTCC 769 compared with other reference strains namely G. hansenii NBRC 14820T. This condition is consistent with the results of the identification of fourth cellulose producing acetic acid bacterial isolates based on phenotypic traits. Thus, the whole cellular protein profiles by SDS-PAGE technique can be used as a one of method to identification of cellulose producing acetic acid bacterial isolates.

GENETIC VARIATION OF MAIZE GENOTYPES (ZEA MAYS L.) DETECTED USING SDS-PAGE

The assessment of genetic diversity among the members of a species is of vital importance for successful breeding and adaptability. In the present study 20 old genotypes of maize from Hungary, Union of Soviet Socialist Republics, Poland, Czechoslovakia and Slovak Republic were evaluated for the total seed storage proteins using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) through vertical slab unit. The number of total scorable protein bands was twentythree as a result of SDS-PAGE technique but those that were not cosistent in reproducibility and showed occasional variation in sharpness and density were not considered. Out of twentythree polypeptide bands, 6 (31%) were commonly present in all accessions and considered as monomorphic, while 17 (65%) showed variations and considered as polymorphic. On the basis of banding profiles of proteins of different kDa, gel was divided into zones A, B and C. The major protein bands were lied in zones A and B, while minor bands were present in zones C. In zone A out of 10 protein bands, 1 were monomorphic and 9 were polymorphic. In zone B out of 8 protein bands, 3 was monomorphic and 5 was polymorphic and in zone C out of 5 protein bands, 2 were monomorphic whereas 3 polymorphic. The dendrogram tree demonstrated the relationship among the forty registered old maize genotypes according to the similarity index, using UPGMA cluster analysis. The dendrogram was divided into three main clusters. The first cluster contained Czechoslovakia genotype Mikulická and the second cluster contained Czechoslovakia genotypes Celchovicka ADQ. Cluster 3 was divided into 3 subclasters. Similarly the present study of genetic variability in the seed storage polypeptide determined by SDS-PAGE technique proved that it is fruitful to identify genetic diversity among accessions of maize.

Genetic variation and relationships of old maize genotypes (Zea mays L.) detected using SDS-page

The assessment of genetic diversity among the members of a species is of vital importance for successful breeding and adaptability. In the present study 40 old genotypes of maize from Hungary, Union of Soviet Socialist Republics, Poland, Czechoslovakia, Yugoslavia and Slovak Republic were evaluated for the total seed storage proteins using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) through vertical slab unit. The number of total scorable protein bands was twentythree as a result of SDS-PAGE technique but those that were not cosistent in reproducibility and showed occasional variation in sharpness and density were not considered. Out of twentythree polypeptide bands, 6 (31%) were commonly present in all accessions and considered as monomorphic, while 17 (65%) showed variations and considered as polymorphic. On the basis of banding profiles of proteins of different kDa, gel was divided into zones A, B and C. The major protein bands were lied in zones A and B, while minor bands were present in zones C. In zone A out of 10 protein bands, 1 were monomorphic and 9 were polymorphic. In zone B out of 8 protein bands, 3 was monomorphic and 5 was polymorphic and in zone C out of 5 protein bands, 2 were monomorphic whereas 3 polymorphic. The dendrogram tree demonstrated the relationship among the forty registered old maize genotypes according to the similarity index, using UPGMA cluster analysis. The dendrogram was divided into two main clusters. The first one contained eleven genotypes from maize, while the second cluster contained the twentynine genotypes of maize. Similarly the present study of genetic variability in the seed storage polypeptide determined by SDS-PAGE technique proved that it is fruitful to identify genetic diversity among accessions of maize.

Numerical analysis of whole-cell and cell wall proteins’ profiles of human oral cavity Candida isolates

Protein electrophoresis has been of great value for the delineation of fungi taxonomy. The aim of this study was to evaluate the protein polymorphism degree among thirteen oralCandida strains belonging to six species and isolated from adult patients submitted to the Dental Hospital of Monastir (Tunisia) and suffering from denture stomatitis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in order to identify the similarities in oral isolates and to establish their possible dissemination route. Proteins were extracted using two different protocols (cell wall and whole-cell proteins) and then submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed using the Gel Pro Analyzer (3.1). Dendrograms of each proteins gel were generated by the unweighted pair group arithmetic average (UPGMA 4.0) software package. A total of 30 major phenons (clusters) were analyzed, according to their homogeneous (species and period) and heterogeneous (distinct species and/or period) characteristics when we analyzed the cell wall proteins profiles and also 30 major phenons of whole-cell proteins patterns. The protein profile of each Candida isolate using two different methods was patient-specific and not associated with the date of isolation or the cell surface hydrophobicity (CSH) of the strain. Key words: Candida, cell wall proteins, whole-cell proteins, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), hydrophobicity.

Phenotypic and genotypic diversity among strains of Aureobasidium pullulans in comparison with related species

Intra-specific diversity of 200 Aureobasidium pullulans strains isolated from different sources and their relatives Kabatiella lini CBS 125.21 T and Hormonema prunorum CBS 933.72 T were studied by assessment of macromorphological, and physiological tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique (SDS-PAGE) of whole-cell proteins as well as enterobacterial repetitive intergenic consensus (ERIC)-, repetitive extragenic palindromic (REP)- and BOX-PCR techniques (collectively known as rep-PCR). Rep-PCR is an efficient procedure for discrimination of A. pullulans in terms of simplicity and rapidity. RFLP-PCR technique was applied for the identification of A. pullulans isolates and distinction from related species. This technique was insufficient for investigation of intra-specific diversity. The tested strains of A. pullulans could be divided into two groups based on their macromorphological, protein patterns obtained after SDS-PAGE as well as rep-PCR patterns. The first group of strains shared similar characteristics and was very different from the second one, designated as "complex group", consisting of strains with very little similarities within the group. Phenetic analysis of ERIC banding patterns failed to group the isolates on the basis of their substrate or geographical origin. Using 18S rDNA gene sequence analysis of selected isolates, three strains: HoHe3 km, A. pullulans DSM 62074 and H. prunorum CBS 933.72 T were distinguished from all other analysed members of genera Aureobasidium and Kabatiella.

Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing.

A new method for on-chip sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins is reported. Miniaturization of SDS-PAGE has attracted significant attention because it offers rapid analysis times, excellent resolution, high throughput, and the potential for integration and automation, as compared to conventional counterparts. The presented on-chip SDS-PAGE technique employed photolithographically patterned, cross-linked gels fabricated in situ in <20 min. The effects of sieving gel composition on the migration properties of fluorescently labeled protein standards (ranging in molecular weight from 14.2 to 66 kDa) were quantified, as was the ability of the gels to function as a sieving matrix for biologically relevant species. Ferguson analysis was employed to calculate retardation coefficients and free solution mobilities. In conjunction with fluorescence imaging, the on-chip SDS-PAGE separation mechanism was evaluated in terms of separation performance indexes, as well as limiting behaviors (i.e., free solution mobility, exclusion characteristics). The photolithographically fabricated gels employed for on-chip SDS-PAGE allowed rapid (<30 s) separations of proteins in short separation lengths (4 mm) with efficiencies as high as 4.41 x 10(5) plates/m. The on-chip SDS-PAGE separations were approximately 100 times faster than conventional slab gel SDS-PAGE (60 min) and occurred in a fraction of the separation length required by slab gels. The migration behavior of protein standards correlated well with molecular weight and allowed molecular weight determination for interleukin-2, fibroblast growth factor, insulin-like growth factor, and tetanus toxin C-fragment.

Identification of lactic acid bacteria from spontaneous fermentation of ‘Almagro’ eggplants by SDS-PAGE whole cell protein fingerprinting

In order to complete the previously performed phenotypic identification [Sánchez et al., 2000. Int. J. Food Microbiol. 59 (2000) 9], whole cell protein patterns obtained using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of 149 lactic acid bacteria (LAB) strains, isolated from ‘Almagro’ eggplants spontaneous fermentation, were analysed. Phenotypic identification of those strains had revealed the presence of the species Lactobacillus (Lb) plantarum, Lb. pentosus, Lb. brevis biotype 2, Lb. brevis biotype 3, Lb. fermentum, Lactobacillus spp. and Aerococcus viridans. The SDS-PAGE technique generated complex and stable patterns that were easy to interpret and compare with those of the 17 reference strains used in this study. After numerical analysis of the resulting electrophoretic protein patterns, six well-delineated clusters were discriminated that, with some exceptions, correlated well with the different groups phenotypically found. Only two strains showed unique protein patterns without meaningful homology to any of the reference strains used for this study and remained unidentified.

A rapid method for identification of Mycobacterium species by polyacrylamide gel electrophoresis of soluble cell proteins

The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of an easily and rapidly prepared soluble protein fraction were used in conjunction with conventional techniques to identify different strains of Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. avium, M. kansasii, M. marinum, M. gastri, M. simiae and M. malmoense. Complete concordance of results from both methods was obtained with all species except those of the M. tuberculosis complex. With the SDS-PAGE technique, all strains of the M. tuberculosis complex were recognised as belonging to one species. By visual analysis of the SDS-PAGE polypeptide profiles, only minor differences between strains of the same species were seen and each species showed a characteristic polypeptide profile. Quantitation of the data by calculation of the Dice coefficient of similarity of the band positions obtained by densitometry indicated that the similarity between different strains of one species was 90-100% and the similarity between the species was in the range 30-45%. The results indicate that SDS-PAGE is a simple and rapid method for identifying mycobacterial strains.

Hemoglobin in the parthenogenetic eggs ofDaphnia magna

The hemoglobin (Hb) concentration in a single parthenogenetic egg from Hb-rich Daphnia magna was about three times that in an egg from a Hb-poor animal. Hemoglobin-rich animals first produced Hb-rich eggs in air-saturated water. The Hb concentration of the eggs gradually decreased, leading to a decrease in the Hb concentration in hemolymph in adult animals. The P50 values (equilibrium oxygen pressure at half saturation) of solutions of purified Hb were 2.9 and 7.2 torr at 20°C for Hb-rich and Hb-poor eggs, respectively. Hemoglobin from the eggs was separated into at least eight Hb components by isoelectric focusing. The pI values (isoelectric point) of the major Hb components of the eggs exceeded those of adult Hb. The relative amounts of Hb components with high pI values in Hb-rich eggs were higher than those of Hb-poor eggs. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique (SDS-PAGE), the Hb from eggs was composed of 37,000 molecular weight subunits.

Heat‐shock proteins during pollen development in maize

AbstractIn contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat‐shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat‐shock responses. One‐dimensional Sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis technique (SDS‐PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat‐shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat‐stress conditions; however, the consti‐tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.

Separation of cardiac myosin heavy chains by gradient SDS-PAGE.

Separation of alpha- and beta-myosin heavy chains (MHCs) in cardiac ventricles of rats by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was accomplished and compared with the separation of myosin isozymes obtained with pyrophosphate gels. Whole muscle homogenates were electrophoresed on a 4-9% linear gradient SDS polyacrylamide gel for 3-4 h. MHC bands were identified by the migration distance relative to a MHC standard and immunoblot results with a monoclonal antibody to MHC. The MHC bands were further identified as alpha and beta based on the electrophoretic mobility of ventricular homogenates from hypothyroid and hyperthyroid rats and ventricular and slow soleus skeletal muscle homogenates from control rats. The beta-MHC migrated faster than alpha-MHC, and laser densitometry revealed separate peaks when both MHCs were present. With homogenates containing MHC ranging from 0 to 100% alpha, the separation of MHCs with gradient SDS-PAGE correlated highly (r = 0.97) with separation of myosin isozymes by pyrophosphate gel electrophoresis. The SDS-PAGE technique reported herein is a quick, valid, and direct method for the identification and quantification of ventricular MHCs.

Genetic control of endosperm proteins in wheat

Total endosperm protein subunits, extracted from the common wheat cultivar Chinese Spring and from some of its aneuploid lines, were fractionated according to their molecular weight (MW) in an improved high resolution one-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). The resolution obtained by this method and, in particular, that of the high molecular weight (HMW) glutenin and gliadin subunits approached that of a previous report in which two-dimensional fractionation system based on charge and MW was used. In the cultivar Chinese Spring, 21 discrete protein bands were resolved and the chromosomes controlling many of them were either reconfirmed, or, in some cases, established. The advantages of this high resolution SDS PAGE technique are discussed.

Polypeptide phosphorylation in adenovirus-infected cells.

Cells infected with human adenovirus type 5 have been labelled with 32P-orthophosphate under various conditions and extracts examined, after denaturation in sodium dodecyl sulphate (SDS), by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. A number of polypeptides appear to be phosphorylated specifically as a result of infection. Early in infection, phosphorylation of a polypeptide of apparent mol. wt. 26 K associated with ribosomes can be detected. Two other phosphorylated polypeptides of apparent mol. wt. 72 K and 18 K can also be seen, the former being mainly confined to the nucleus and capable of being precipitated by the previously described virus-specific P antiserum. The 18 K phosphorylated polypeptide is found mainly in association with membrane fractions. Later in infection phosphorylated polypeptides of apparent mol. wt. 100 K and 39 K can be recognized, the former being associated with ribosomes but removed, however, with a high salt wash; the latter component is mainly detected in the nucleus. Analysis of the purified 32P-labelled virus by the SDS PAGE technique indicated that a structural polypeptide of apparent mol. wt. 66 K (IIIa) was also phosphorylated.