Gel Electrophoresis Apparatus Research Articles

Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03–10 μg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.

Electrophoretic Pattern of Serum Proteins in Pregnancy: A Cross-sectional Study

Introduction: Nutritional status during pregnancy is a determining factor for maternal and neonatal health outcomes. Protein is an essential nutrient for foetal development. If pregnancy is associated with an improper diet leading to protein deficiency, the prospects for maternal and foetal outcomes may be catastrophic. An electrophoretic study of serum protein patterns in pregnancy can help identify pregnancy-specific protein markers and facilitate the monitoring of feto-maternal health. Aim: The aim of this study is to assess serum protein levels and their fractions (albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin) using gel electrophoresis in healthy pregnant women attending an antenatal clinic. Materials and Methods: This cross-sectional study was conducted in the Department of Medical Biochemistry in collaboration with the Department of Obstetrics and Gynecology at Sultania Zanana Hospital, associated with Gandhi Medical College, Bhopal, Madhya Pradesh, India, between January 2021 and February 2021. Fifty normal non-pregnant women of childbearing age and 50 normal pregnant women in the first trimester of pregnancy attending an antenatal clinic were enrolled and followed-up through the second and third trimesters for investigations. Serum total protein, albumin, and globulin levels were estimated using a fully automated chemistry analyser, and the electrophoretic patterns of serum protein fractions were analysed using an automated gel electrophoresis apparatus. EpiInfo statistical software was used for the statistical analysis of data, with p<0.05 considered the statistical significance level. Results: The serum total protein level in non-pregnant women was 6.36±0.40 g/dL, while in pregnant women, it was 8.01±0.40 g/dL, 8.00±0.42 g/dL, and 6.36±0.43 g/dL in the first (T1), second (T2), and third (T3) trimesters, respectively. The serum albumin level was 3.90±0.30 g/dL in non-pregnant women, while in pregnant women it was 4.24±0.38 g/dL, 4.58±0.34 g/dL, and 3.87±0.36 g/dL in T1, T2, and T3, respectively. The globulin level in non-pregnant women was 2.46±0.28 g/dL, while in T1, T2, and T3 it was 3.86±0.22 g/dL, 3.42±0.20 g/dL, and 2.49±0.24 g/dL, respectively. A significant difference was found among the groups for serum total protein, serum albumin, and serum globulin. In non-pregnant women, the protein fractions- albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulindid not show any deviation from normal levels. However, there was a decrease in albumin and beta globulin levels, along with an increase in alpha-2 globulin levels in the first trimester. An increase in beta globulin and a decrease in gamma globulin levels were observed in the second trimester. In the third trimester, all these fractions did not show any significant alterations, except for gamma globulin. Conclusion: Serum total protein, serum albumin, and serum globulin levels showed a significant difference. However, a slight increase in serum globulin levels compensated for the fall in albumin levels during pregnancy. Variations in the levels of beta globulin, alpha-2 globulin, and gamma globulin were observed during pregnancy, while, on the other hand, alpha-1 globulin did not show any alterations in pregnant women during the first, second, and third trimesters, although its level was higher in pregnant women compared to non-pregnant women.

High-resolution nucleic acid detection using online polyacrylamide gel electrophoresis platform

Polyacrylamide gel electrophoresis (PAGE) is one of the most popular techniques for the separation and detection of nucleic acids. However, it requires a complicated detection procedure and offline detection format, which inevitably leads to band broadening and thus compromises the separation resolution. To overcome this problem, we developed an online PAGE (OPAGE) platform by integrating the gel electrophoresis apparatus with the gel imaging system, so as to obviate the need for the complicated detection procedure. Notably, OPAGE enabled the real-time monitoring of the separation process and the immediate imaging of the separation results once the electrophoresis ended. Using a series of synthetic DNAs with different lengths as samples, we demonstrated that the OPAGE platform enhanced 32–64 % of the number of theoretical plates, showed a robust dynamic range of 0.1–12.5 ng/μL, and realized a limit of detection as low as 0.08 ng/μL DNA. Based on our results, we anticipate that the OPAGE platform is a promising alternative to traditional nucleic acid gel electrophoresis for simple and high-resolution detection and quantification and nucleic acid.

A simple and fast triplex-PCR for the identification of milk's animal origin in Halloumi cheese and yoghurt.

In this research communication we describe a straightforward triplex-PCR protocol able to differentiate the origin of milk from three closely related species (goat, sheep and cow) in Halloumi, a cheese with Protected Designation of Origin (PDO), and yogurts. Halloumi must contain at least 51% sheep or goat milk, therefore, the fraudulent adulteration of this cheese with excess of cow milk must be routinely tested. The assay employs one universal forward primer and three species-specific reverse primers giving rise to 287 bp (cow), 313 bp (goat), and 336 bp (sheep) amplicons, under the same amplification conditions. This protocol, when used to test a small number of Cyprus commercial products, correctly detected mislabeling in Halloumi (2 out of 6 samples were adulterated) and yogurt brands (1 out of 4 was adulterated). The suggested protocol is a reliable tool for identifying the origin of milk in Halloumi cheeses and yogurts and can be used in any laboratory equipped with a thermocycler and an agarose gel electrophoresis apparatus.

On-Site Genomic Epidemiological Analysis of Antimicrobial-Resistant Bacteria in Cambodia With Portable Laboratory Equipment.

The rapid emergence of carbapenemase-producing gram-negative bacteria (CPGNB) is a global threat due to the high mortality of infection and limited treatment options. Although there have been many reports of CPGNB isolated from Southeast Asian countries, to date there has been no genetic analysis of CPGNB isolated from Cambodia. Sequence-based molecular epidemiological analysis enables a better understanding of the genotypic characteristics and epidemiological significance of antimicrobial-resistant (AMR) bacteria in each country, and allows countries to enact measures related to AMR issues. In this study, we performed on-site genomic epidemiological analysis of CPGNB isolated in Cambodia using a portable laboratory equipment called Bento Lab, which combines a PCR thermal cycler, microcentrifuge, gel electrophoresis apparatus, and LED transilluminator, along with the MinION nanopore sequencer. PCR targeting of major carbapenemase genes using Bento Lab revealed that two Escherichia coli isolates and one Acinetobacter baumannii isolate harbored carbapenemase genes: blaNDM, blaOXA–48, and blaOXA–23, respectively. The results of phenotypic diagnostic tests for CPGNB, such as the carbapenem inactivation method and double-disk diffusion test using a specific inhibitor of metallo-β-lactamases, were consistent with their AMR genotypes. Whole-genome sequencing analysis using MinION revealed that blaNDM–5 gene was carried on a 93.9-kb plasmid with IncFIA/IncFIB/IncFII/IncQ1 replicons, and blaOXA–181 gene was carried on a 51.5-kb plasmid with the IncX3 replicon in E. coli isolates. blaOXA–23 was encoded in two locations on the chromosome of A. baumannii. Plasmids carrying blaNDM–5 or blaOXA–181 in E. coli were highly structurally identical to plasmids prevalent in Enterobacterales in China and other countries, suggesting that they disseminated from a common evolutionary origin. Our findings demonstrate the potential impact of portable laboratory equipment on AMR bacteria research in hospitals and research centers with limited research facilities, and provide the first glimpse into the genomic epidemiology of CPGNB in Cambodia.

Development of a do-it-yourself (D.I.Y.) gel electrophoresis apparatus for Grade-12 STEM general biology students

A hurdle to effective science education in schools is often the lack of teaching materials. Teaching science education in the 21st century requires direct visualization and manipulation with effective use and implementation for meaningful learning to occur, thus, unleashing students’ potential towards the lesson, boosting their engagement in learning, and applying the knowledge into skills. This study was carried out to develop a do-it-yourself gel electrophoresis apparatus. This study was conducted in Cebu Normal University, Philippines with nineteen (19) teachers from the Methods of Teaching Science and Instructional Trends and Issues Class who evaluated the apparatus in terms of usability and functionality. The respondents tested the Gel Electrophoresis apparatus by conducting the experiment and they evaluated the apparatus’ functionality and usability by using an evaluation sheet which is composed of eleven (11) usability questions, four (4) functionality questions. Suggestions were also asked to improve the said apparatus. After gathering data, the suggestions made by the respondents were incorporated which resulted to the improved D.I.Y. Gel Electrophoresis Apparatus. A manual with the procedures was also designed to further guide students and teachers on how to build the apparatus. With the findings and results obtained in the study, the respondents recommended the usage of the developed do-it-yourself gel electrophoresis apparatus in their respective classes and agreed that it is a good source of learning the concept of gel electrophoresis as a 21st century biological technique. The apparatus was easy to set-up and is of low-cost. During experimentation, the apparatus yielded with a positive result – the separation of food dyes which follows the principle of gel electrophoresis. Moreover, the experiment encourages creativity and it helps with the understanding of the nature of DNA and the concept of gel electrophoresis as a 21st century biological skills.

Genetic characterization and assessment of diversity in Pandharpuri buffalo breed of India using heterologous microsatellite markers

The present study was aimed at genetic characterization and diversity analysis of Pandharpuri buffalo population using the Food and Agriculture Organization (FAO) recommended bovine microsatellite markers. Genomic DNA was isolated from blood samples of 50 unrelated animals and a total of 23 microsatellite loci were amplified using polymerase chain reaction. Among 23 recommended microsatellite markers, 14 markers (BM2113, BM1818, CSSM66, HEL13, INRA037, ILSTS05, HAUT27, INRA023, INRA035, HEL5, ETH3, NRA063, MM12 and ETH10) were found to be highly polymorphic in Pandharpuri population. The amplified products were run on polyacrylamide gel electrophoresis (PAGE) apparatus along with a ladder; subsequently, the allele typing was done based on silver staining of the gel. The effective and observed number of alleles, heterozygosity (expected and unbiased) estimates and polymorphic information content (PIC) levels were estimated for each locus. A total of 87 alleles were secured for 14 polymorphic loci studied giving an overall average of 6.21 alleles per locus. The number of alleles ranged from 2 (INRA063) to 9 (BM1818 and HEL13). The mean effective number of alleles across all polymorphic loci was found to be 4.28. The overall mean expected heterozygosity and unbiased expected heterozygosity values were 0.77 and 0.76, ranging from 0.50 (INRA063) to 0.88 (BM1818) and 0.50 (INRA063) to 0.88 (BM1818), respectively. The average PIC estimate across all polymorphic loci was 0.73 ranging from 0.373 (INRA063) to 0.864 (BM1818). In the present study, the characterization and diversity estimates are reported for Pandharpuri population. It was found to maintain optimum diversity based on 14 polymorphic microsatellite markers.

A crude method of DNA extraction and identification from exfoliated human buccal mucosa cells.

DNA analysis has a key role in forensic dentistry. However, techniques of DNA extraction and analysis are far from the reach of majority of medical professionals owing to its expensive set up. The present study was aimed at formulating a crude method of extracting DNA from human buccal mucosa cells using materials commonly available in the laboratory so that the medical professionals could get more exposure to molecular biology techniques. The objectives were to identify the DNA and to assess its purity. Buccal mucosa cells from 10 healthy volunteers were taken for DNA extraction following the protocol of cell lysis, purification, and precipitation. DNA was identified using standardized techniques like Diphenylamine test and its purity was assessed using a spectrophotometer. A gel electrophoresis apparatus was also constructed using readily available materials. DNA was extracted from human buccal mucosa cells using a crude method. The standardized tests confirmed the presence of DNA contaminated with proteins. The locally made Gel electrophoresis model exhibited a faint halo around the wells instead of DNA bands. DNA extraction from human buccal mucosa cells was made possible using locally available materials and a crude method, but it was not of high purity.

Two-Dimensional Gel Electrophoresis: Vertical Isoelectric Focusing.

Two-dimensional gel electrophoresis is one of the most powerful tools for separating proteins based on their size and charge. Two-dimensional gel electrophoresis (2-DE) is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

A Multichannel Gel Electrophoresis and Continuous Fraction Collection Apparatus for High-Throughput Protein Separation and Characterization.

We developed a multichannel gel electrophoresis system that continuously collects fractions as protein bands migrate to the bottom of gel columns. The device uses several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "counter-free-flow" elution technique allows continuous and simultaneous fraction collection from multiple channels at low cost. Using the system with SDS-PAGE, 300μg samples of protein can be separated and eluted into 48-96 fractions over a mass range of 10-150kDa in 2.5h. Each eluted protein can be recovered at 50% efficiency or higher in ~500μL. The system can also be used for native gel electrophoresis, but protein aggregation limits the loading capacity to about 50μg per channel and reduces resolution. This system has the potential to be coupled with mass spectrometry to achieve high-throughput protein identification.

A tetrad apparatus for protein gel casting, electrophoresis, staining, and scanning techniques with dual sensors for automatic detection of gel polymerization and protein migration.

Recent advancements in biochemical sciences have facilitated researchers to explore the structure and function of macro molecules in a cell. PAGE is one of the most favored and adapted laboratory techniques. Due to its simple and economical procedures, several variants or new modifications are routinely observed in the basic electrophoresis technique that comprises gel casting, electrophoresis, staining, and imaging process which consequently necessitates additional apparatuses/components in the laboratory. Operation of these additional apparatuses/components lengthens the pre- and postelectrophoresis procedures involving many intermittent tedious and time-consuming steps. A universal apparatus that can facilitate all such associated techniques is lacking and is of utmost importance for fast and effective results. An apparatus that can perform synchronized action of slab gel casting (16×16cm), electrophoresis (SDS-PAGE), dye staining (Coomassie), and imaging (scanning) techniques with real-time monitoring through sensor technology is described in this article. The estimated cost (∼$150) of fabrication of the apparatus is very economical and simple assembly procedure of the main apparatus can be completed within ∼30min after fabrication.

Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction.

This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP), and LAMP-Lateral Flow Dipstick (LFD). For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s). After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required.

Electrophoresis-staining apparatus for DNA agarose gels with solution exchange and image acquisition

ABSTRACTAgarose gel electrophoresis is routinely used for the separation of nucleic acids. Gels are developed, stained, and visualized using dedicated equipment and reagents. Manufacturers have developed instrumentation with advanced features that provide good safety and user-friendly operation. However, the process of size fractionation of nucleic acids by horizontal gel electrophoresis by dye staining may be cumbersome and unsafe due to many steps and harmful chemicals. Here is reported a safe, inexpensive, time-saving, and comprehensive apparatus for gel electrophoresis, staining, and imaging. This newly modified apparatus has simple operation and uses existing equipment and off-the-shelf components for easy construction in the laboratory. The apparatus has been shown to perform agarose horizontal gel electrophoresis and associated techniques with ease and simplicity.

Analysis of pork meat using loop mediated isothermal amplification (LAMP) to confirm halal status

A method has been developed for identifying species from pork samples by loop-mediated isothermal amplification (LAMP) analysis of the wild boar (Sus scrofa) mitochondrial DNA D-loop region. DNA was extracted from raw and cooked pork and beef meat and subjected to polymerase chain reaction (PCR) and LAMP. Approximately 185 base-pair PCR products were found in the pork meat samples after amplification of the D-loop region. LAMP products only showed a smear band with the pork samples. Moreover, by adding CuSO4 to the LAMP product, the white Cu(OH)2 precipitate, which formed a ring-shaped deposit, was not seen in the pork samples, but was seen in the beef and negative control samples. Our finding showed the LAMP technique to be a powerful molecular-biological method for species identification. The addition of CuSO4 to the LAMP product is an alternative method that can be used in field studies, and does not require any gelelectrophoresis apparatus.

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments

A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of approximately 10-150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution.

A Simpler and Faster Version of Two-Dimensional Gel Electrophoresis Using Vertical, Mini SDS-PAGE Apparatus

We have modified one of the most useful methods of protein separation; namely, two dimensional gel electrophoresis (2-DE). This modified version of 2-DE is not only simpler and easier but also faster than all the currently available methods. In this method, isoelectric focusing is carried out in the first dimension using a vertical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) apparatus. Following focusing, each individual lane is excised from this gel and after a 90° rotation, is inserted into a vertical specially fabricated SDS-PAGE gel for the second dimension run, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This modified version of IEF can be run in less than 2 hours compared to the overnight run required by the O’Farrel method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus and up to 10 samples can be run simultaneously using one gel. We have analyzed human sera, animal saliva, eye and liver tissue samples to verify this method.

Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

264 SUITABILITY OF BOVINE SEXING PRIMERS FOR SEX DETERMINATION OF CAPRINE CELLS

Recent studies on PCR-based sex determination of caprine cells use amplification of a single-copy Y-chromosome-specific sequence. Our study investigated the suitability of widely used bovine sexing primers for their usefulness in sexing caprine cells. The common use of these sexing primers is to amplify BRY.4, a Y-specific sequence found in bovine cells (Reed et al. 1995 U.S. Pat# 5459038). The major advantage in amplifying these sequences is that they are multiple repeated DNA sequences. This enhances the sensitivity for use on as few as one cell. Early in their use, a problem was discovered amplifying BRY.4 for bovine sexing; one or more copies of a homologous repeat sequence were present in female cells. Copies in female cells differed by the presence of a Hinf1 restriction site. By digesting the DNA with Hinf1 either prior or subsequent to PCR amplification, it was possible to eliminate the troublesome sequence. Our study investigated whether a site homologous to theY-specific sequence was also present in female caprine cells and, if so, it too contained the Hinf1 restriction site, making possible the elimination of the troublesome sequence. The components of the PCR reaction mix were: buffer (50 mm Tris, 1% dextran T-500, 50 mm KCl, 2.5 mm MgCl2, and 0.035% 2- mercaptoethanol), deoxyribonucleotides (5 µm) (Boehringer Mannheim, Basel, Switzerland), primers (5´-GAACTTTCAAGCAGCTGAGGC-3´ and 5´-GATTGTTGATCCCACAGAAGG-3´) (2.5 µm) (custom synthesis, IDT Inc, Coralville, IA, USA), and Taq polymerase (2.7 U) (AmpliTaq DNA Polymerase, Stoeffel Fragment, Perkin Elmer, Branchburg, NJ, USA). Lymphocytes from one male and three female goat species (Saanen, Nubian/Alpine mix, and Albino) were used as the source of DNA. Approximately 100 cells/2 µL were used to ensure the amplification of the sequence homologous to theY-repeat. The DNA replication was carried out using a Corbett Rapid Thermocycler (Model FTS-IS, Corbett Research, Montlake, Australia) in 20-µL volumes, and all assays were run with positive and negative DNA controls. After replication, some samples were digested with Hinf1 (20 U) (New England Biolabs, Ipswich, MA, USA) for 2 h at 38�C. A 3% agarose gel was run in an agarose gel electrophoresis apparatus (Model 52000, IBI-Shelton Scientific, Peosta, IA, USA) for 30 min at 180 volts. All caprine species samples were run in duplicate. The DNA from three female undigested samples banded at the same position as the male samples (130 bp). The DNA from all three female digested samples banded at two positions (50 bp and 80 bp), both lower than the male samples. The male DNA remained unaltered after the Hinf1 digest, which suggests that the Hinf1 site is not present in the amplified DNA from male cells. The assay generated strong sexing bands with as few as 6 male lymphocytes, the lowest number of cells tested. These primers seem suitable if used in conjunction with Hinf1 digestion.