...We have developed a new method utilizing affinity capillary electrophoresis (ACE) for the determination of binding stoichiometries in biochemical systems. Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak corresponding to the free ligand in the resulting electropherogram provides a criterion of binding of a ligand to its receptor protein. For both low (fast off rates) and high (slow off rates) affinity systems, analysis of the integration of free ligand peak in electropherograms as a function of the total concentration of a ligand in samples at constant concentration of receptor protein yields the binding stoichiometry of the ligand to the protein. Applications of this technique to studies of (i) the inhibition of carbonic anhydrases (CA, EC 4.2.1.1, from human and bovine erythrocytes) by 4-alkylbenzenesulfonamide 1, (ii) the interaction of a monoclonal antibody to human serum albumin (anti-HSA) with its antigen HSA, and (iii) the binding of streptavidin (from Streptomyces avidinii) to biotin derivatives (monobiotinylated oligodeoxyribonucleotide 2, fluorescein biotin, or Lucifer Yellow biotin) yield stoichiometries of 1:1, 1:2, and 1:4, respectively. For multivalent, tight-binding systems, this ACE method can readily separate stable intermediate species. This method is generally applicable to both tight- and weak-binding systems, requires only nanograms of proteins and ligands, involves no radioactive materials...
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...Session 4: Agarose Gel Electrophoresis 2. Explain how DNA is able to migrate through the agarose gel and how the charges affect this process? In gel electrophoresis, samples to be separated are applied to a porous gel medium made of a material such as agarose. Once all the samples have been loaded into the wells, the chamber is connected to a power supply and an electrical current (usually 50–150 V) is applied to the gel. The chamber is designed with a positive electrode (anode) at one end and a negative electrode (cathode) at the other end. Electrophoresis literally means “to carry with electricity;” once the electric field is established, charged molecules in the samples migrate through the pores of the gel toward their pole of attraction. Molecules with a net negative charge migrate toward the positive electrode and molecules with a net positive charge migrate toward the negative electrode. The overall charge of a molecule affects the speed at which it travels through the gel. Highly charged molecules migrate more quickly through the gel than weakly charged molecules. The mobility of a molecule during gel electrophoresis also depends on its molecular size and shape. The small pores of the gel matrix act as a sieve that provides great resolving power. Small molecules maneuver more easily through the pores than larger molecules and therefore travel relatively quickly. Large molecules encounter more resistance as they make their way through the tiny pores...
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...norfolkhornbreeders.co.uk/history.php I Protocol To perform a quick screen using agarose gel electrophoresis, you collected some cells from the lambs and the potential parents in order to extract and process the DNA ready for electrophoresis. You labeled your tubes as followed: Sample A: Ladder Sample B: Suffolk ewe Sample C: Lamb 1 Sample D: Lamb 2 Sample E: Norfolk Horn ram Sample F: Southdown ram II DNA electrophoresis procedure Read the procedure carefully before you start. well | 1 | 2 | 3 | 4 | 5 | 6 | Sample | A | B | C | D | E | F | Use a plastic pipette to pour some buffer near the comb. GENTLY remove the comb and drop some more buffer in the wells. Using a micropipette, load 20l of each of the samples into separate wells in the agarose gel as indicated in the above table. Do not stab the pipette tip into the wells and pierce the bottom of the gel! When all samples have been loaded onto the gels, place them into the tank. Call a demonstrator to assist you with covering the gels with the buffer. Cover the tank with the lead and connect the electrodes to the electrophoresis power pack the correct way! DNA will migrate towards the positive electrode, which is usually coloured red. Press the RUN button. The gels will be run at 150 volts for about 30 minutes. TURN OFF THE ELECTROPHORESIS POWER PACK. Switch off at...
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...S | P | X | T (a lot)S | P | X | ROW D | X | P | H (a few) S | X | P | H (a lot) S | 1: The best condition for tetragonal lysozyme crystal formation is Row C in the 5% to 7% NaCL at pH 4.5 (60mg/ml) large crystals 2: The best condition for hexagonal lysozyme crystal formation is Row D in the 2% to 3% NaNO3 at pH 7.5 (60mg/ml) large crystals 3: Tetragonal lysozyme crystals are primarily found in the NaCL solution 4: Hexagonal lysozyme crystals are primarily found in the NaNO3 solution 5: Temperature proved to have a substantial effect on crystal size as all the crystals formed while incubated at 4 degrees Celsius were small crystals. 1HEW Crystal structure. Experiment 4: Polyacrylamide gel electrophoresis (Analysis of proteins) M | 1 | 2 | 3 | M | 4 | 5 | 6 | | 1. * Impure YFP can be seen in column 1 ranging from 70-10 kDa (binding buffer) * Everything except YFP can be seen in column 2 also ranging from...
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...Hope Hayman DNA Bar Coding: Is Convenient and Accurate for Taxonomy Studies Introduction— Different samples of “wild card” subjects and given subjects (Drosophila melanogaster and the Coquina clams) genomic DNA were acquired and isolated through several steps to amplify the cytochrome c oxidase 1 (CO1) genes. Through comparisons of these wild cards and given subjects mitochondrial CO1 genes with the BLAST library, it was revealed that DNA bar coding is convenient and accurate for taxonomy study. DNA bar coding utilizes the amplification and purification of a specific region of the mitochondrial genome by polymerase chain reactions (PCR). DNA bar coding then uses the PCR products ran in gel electrophoresis to analyze. Materials and Methods— DNA Isolation The wild card specimens were obtained and brought to the lab for DNA isolation. The first step in DNA isolation was to lyse the specimen. The specimens were first homogenized individually in 1.5 mL microtubes. 20 µL of proteinase K was added to each homogenized sample. 200 µL of AL buffer was then added to each sample. The samples were vertexed and incubated at 70oC for 10 minutes. After the incubation, 200 µL of 100% ethanol was added and vortexed again. The next step in DNA isolation is to bind the DNA. The lysate was transferred to a spin column and centrifuged at 8000 rpm for 1 minute, and the flow through was discarded. 500 µL of AW1 buffer was added, then centrifuged at 8000 rpm for 1 minute, and the flow through was...
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...discuss three researches done on a prey’s evolution to survive against predation. I will discuss the tail autonomy in Tokay gecko (Sanggaard et al. 2012), the odorous and non-fatal secretion in Wrinkled frog (Yoshimura and Kasuya 2013), and the avoidance response in Brownbanded bamboo shark embryos (Kempster et al. 2013). Together, these studies show the effectiveness and vital impact of evolution to the survival of prey. Sanggaard et al. (2012) researched evolution in Tokay gecko (Gekko gecko) to investigate the mechanism of their tail autonomy by facilitating autonomy. The geckos were euthanized by pentobarbital and the tails were removed by induced autonomy. Moisture from the exposed end of the tail was collected and subjected to Gel Electrophoresis and Mass Spectrometry and analyzed using MS-BLAST. The tails’ structure was analyzed through multiple methods. Through Magnetic Resonance Imaging, a Micro Imaging 5 probe, a Great 60 Imaging system, a saddle coil, and a gradient cooling temperature of 20°C was utilized. Electron Microscopy and 3D Fast Low-Angle Shot sequence were utilized. Scanning Electron Microscopy was utilized and the tails were observed through a FEI NOVA NanoSEM 600. The analysis showed that there was a fat layer around the vertebra and the muscle was between the fat layer and dermis. The autonomy septum divided the fat and muscle regions in each fracture planes. A dorsal median septum, a ventral...
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...food and folk medicine (Hamissou, Smith, Carter, and Triplett 641). Researchers have found that the plant protein inhibits H1N1, H3N2 and H5N1 subtypes. Of the medicinal plants studied, the Momordica Charantia plant has been reported to contain many antiviral properties (Pongthanapisith, Ikuta, Puthavathana, and Leelamanit 1). The seed of the M. Charantia was purified of the protein using Fast Protein Liquid Chromatography. The proteins are separated using Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The technique involves placing the protein mixture on gel containing an immobilized pH gradient. The gel slows the passage of proteins and acts as a molecular filter. Samples are loaded into the SDS-polyacrylamide gel and the electric field is applied. The gel slows the passage of proteins and acts as a molecular filter separating different protein molecules according to their size. Smaller proteins move faster than larger proteins and are found near the bottom of the gel. The gel is treated with a coomassie blue stain that binds to the proteins and allows the researcher to visualize the protein bands. The SDS-PAGE method is better suited for separating smaller molecules like protein.(Nelson and Cox 93-94) Madin-Darby canine kidney cells were inoculated with Influenza virus and incubated. The number of infected cells was counted under a microscope. Proteins of the Momordica Charantia were added to the infected cells. After further incubation overnight, the infected...
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...LATEST BIOCHEMICAL TECHNIQUES CAPILLARY ELECTROPHORESIS Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom’s radius. In conventional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. The rate at which the particles moves is directly proportional to the applied electric field; the greater the field strength, the faster the mobility. If two ions are the same size, the greater charge will move the fastest. For ions of the same charge, the smaller particle has less friction and overall faster migration rate. The technique of capillary electrophoresis was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. Capillary electrophoresis is used most predominately because it gives faster results and provides high resolution separation. It is a useful technique because there is a large range of detection methods available. PRINCIPLE Electrophoresis is the process whereby the movement of ions is produced under the influence of an applied voltage across a field that the ions exist. In electrophoresis, ions that are negatively charged will move or migrate towards the positively charged electrode while ions that are positively charged will...
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...also called sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique used to separate proteins only by their size. SDS or sodium dodecyl sulfate is a detergent used to denature the proteins; they will unfold and will not have any secondary, tertiary or quaternary structure. Proteins will have a negative charge and they will move down the gel by their molecular weight. PAGE or polyacrylamide gel electrophoresis is a polymer of the neurotoxin acrylamide and a cross-linking agent called bis-acrylamide. Usually APS and TEMED are used as catalysts for the process of cross-linking. There are two different settings of running the polyacrylamide gel, denaturing gel and non-denaturing gel or native gel. Denaturing gel consists of using urea or SDS to denature or unfold proteins and subsequently separate them by their molecular weight. A non-denaturing or native gel is used primarily to analyze the tertiary and quaternary structure of proteins; therefore they do not need to be unfolded. SDS-PAGE is essential in protein analysis in different fields such as forensic, biochemistry, biotechnology, molecular biology and genetics. The SDS-PAGE gel has to be poured in two parts. The first gel that is poured is the resolving gel. This type of gel consists of 3 to 30 % acrylamide thus possessing small pores and a pH of 8.8. The second gel is called the stacking gel and it is poured on top of the resolving gel. Stacking gel has 8 to 12 % acrylamide, therefore possessing...
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...Introduction IgG is an antibody probe that has an affinity for binding specifically to the IgG protein antigen. The research goal was to identify how closely the IgG of the tested species are related. This was accomplished by examining how goat IgG, specific for goat protein, responded to the same protein from cows, pigs, rats, and humans, and it was determined which animals have the most similar antigen binding reactions. The variability in each animal’s IgG protein was revealed by the level of affinity that the anti-goat IgG probe had for the species tested. Since the variability in each animal’s proteins is due to their genetic coding sequence this study clarified which species have the most similar genetic code. Immunoglobulins are the antigen-recognition molecules of B cells and are used as the main effector function in adaptive immunity. (Janeways, 2001) Their are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM and all five Ig classes are present in mammals and are produced from B lymphocytes as part of the immune response system. (Urich, 1994) IgG antibodies are large molecules, having a total molecular weight of 150kDa, composed of two heavy (H) peptide chains weighing approximately 50kDa and two light (L) peptide chains approximately 25kDa. (Janeways, 2001). The region of Light and Heavy chains connected by a disulfide bond makes up the Fragment antigen binding (Fab) structure, while the remaining Heavy chain region is referred to as the...
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...Figure 1: Agarose Gel Electrophoresis depicting the PCR product and genomic DNA of fruit flies This agarose gel image was used to visualized the DNA sequence that was isolated from the fruit flies. Agarose Gel Electrophoresis results from Section 3 Bench 5. EGFR was used as the primer RTK gene for this set and sequence. This is the PCR product. Lane 1, ladder; Lane 2, Genomic DNA and it contains the long streak of DNA; Lane 3, Uncut Plasmid; Lane 4, Linear Plasmid that was digested by EcoRI at 3kb; Lane 5, PCR 1; Lane 6, PCR 2 and these lanes (4-6) 9 show how the specific inserts were applied during PCR; Lane 7, Combined PCR that was purified during miniprep; Our target insert was shown in these lanes at 1 kb too. Lane 8, Ladder. This figure shows...
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...study the principle of gel electrophoresis. 2. To use gel electrophoresis method to measure the nucleic acid solutions. 3. To learn the technique of nuclei acid measurement by using gel electrophoresis. Introduction: Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA. The components of gel electrophoresis system is power supply and a chamber, agarose gel which is a porous material that allows molecules migrate through, buffer with a mixture of water and ions, and gel casting tray and comb. Electrophoresis is a method of separating substances based on the rate of movement while under the influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose gel is created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, and then pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab. Nucleic acids are composed of chains of nucleotides. The ‘backbone’ of the nucleic acid structure is a repeating chain of phosphate groups and pentose sugars. At certain pH values, the oxygen atoms in the phosphate groups ionize, giving the molecule an overall negative charge. If exposed to an electric field, these molecules are attracted to the positive terminal. During electrophoresis, the gel is submersed in a chamber...
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...I. 진단혈액 진단혈액 수련항목 (1) : 혈액학적검사 기본 술기 표준수련기간 : 1주 수련내용 : ◆ 용어정의 : • 혈액학적검사 : 혈액세포와 응고에 관련된 일련의 검사를 의미한다. 혈구의 체내분포, 구조, 기능에 관련된 검사, 골수에 분포하는 전구세포에 관한 검사, 혈구에 영향을 끼칠 수 있는 혈장 인자에 관한 검사 및 유전자 이상에 관련된 검사 등을 포괄적으로 포함한다. • 망상적혈구수 : 적혈구 성숙 단계 중 정염색성 적아구(orthochromatophilic normoblast) 바로 다음 단계의 세포로 핵이 빠져나간 직후부터를 의미한다. 미토콘드리아, 중심소체(centriole), 리보솜 등을 함유하고 있으며 말초혈액에서 24-48시간의 성숙과정을 거쳐서 성숙한 적혈구로 된다 (Ref. Williams 16th p373-374) ◆ 숙지할 필수 지식 : • 혈액학 검사에 사용되는 검체와 항응고제의 작용기전 및 종류 • 모세관 혈액의 채취 방법과 용도, 채취 시 주의점 및 정맥혈과의 차이점 • 적혈구침강계수(ESR) 검사의 원리 ◆ 습득할 필수 술기 : • Neubauer chamber의 사용 • 미량법(micromethod)를 이용한 헤마토크리트의 측정 • 수기법을 이용한 망상적혈구수 검사 ◆ 국내외 장비 및 시약 현황 : 해당없음 ◆ 추천되는 참고자료 : • 대한혈액학회. 혈액학, 2006. • 대한진단검사의학회 편, 진단검사의학 제 3판, 2001. • Henry, JB. Clinical Diagnosis and Management by Laboratory Methods, 24th ed. 2006. 보고서 제출 일자 : 200 년 월 일 평가자 : 지도전문의 인 (일자 : 200 년 월 일) 과장 인 (일자 : 200 년 월 일) 수련위원 인 (일자 : 200 년 월 일) 진단혈액 수련항목 (2) : 자동 혈구계산기 표준수련기간 : 2주 수련내용 : ◆ 용어정의 : • 헤마토크릿(Hct) : 혈액 전체 부피에 대한 적혈구 부피의 비율, 단위는 % 또는 L/L • 평균적혈구용적(MCV) : 적혈구의 평균 용적, 단위는 fL, • MCV = Hct (L/L) X 1,000/RBC count (X1012/L) • 평균적혈구혈색소(MCH) : 적혈구 한 개당 혈색소 양, 단위는 pg, • MCH = hemoglobin (g/L)/RBC count (X1012/L) • 평균적혈구혈색소농도(MCHC) : 적혈구 한 개당 평균 혈색소 농도, • 단위는 g/L, MCHC = hemoglobin (g/L)/Hct (L/L) • 적혈구분포지수(RDW)...
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...DNA Introduction Large pieces of DNA can be broken up into smaller fragments by bacterial enzymes known as restriction endonucleases (RE). They bind to specific nucleotide sequences within DNA, known as recognition sequences/sites and cleave the phosphate backbone at a point within this sequence. ------------------------------------------------- The recognition sequences are usually fairly short (4-8 base pairs) and have two characteristics: 1) They are PALINDROMIC i.e. the sequence on one of the DNA strands is repeated in reverse on the other e.g. 5'...GAATTC...3' 3'...CTTAAG...5' | 2) They are cleaved by REs in one of two ways, either producing blunt ended fragments, or overhangingfragments | | Since there are only 4 bases in DNA, the probability that a particular cut site is found in a random piece of DNA can be quite high. This is particularly true if the site contains only two bases e.g C and G and the DNA has a higher number of G/C pairs than A/T pairs. In this situation more fragments will be produced. Plasmids are circular pieces of DNA found in bacteria and are frequently used in laboratories to house "foreign" genes. The ability to selectively cut plasmids with REs is extremely useful in genetic engineering. The aim of this experiment is to digest two plasmids, one normal (plasmid 1), one mutant (plasmid 2) and analyse the results to determine the nature of the mutation. Digesting plasmid DNA with restriction...
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...Results of Gel Electrophoresis For the gel electrophoresis of the genomic DNA, a band of genomic DNA (roughly 23kbp) is observed near the top of the gel. Further down the gel is another block of bands (ranging from 100bp ~ 2000bp) which is the degraded RNA. My group’s sample (lanes 16&17) shows similar band patterns to that of other groups where genomic DNA and RNA bands are seen but in differing quantities of DNA since some bands appear more faint than the others. In the gel electrophoresis of the plasmid DNA, 4 distinct bands of DNA are observed. The top band of roughly 23kbp is the nicked/relaxed circular plasmid. Below it is another band of roughly 9500bp of linear plasmid DNA. The following band of around...
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