electrophoresis presentation

Electrophoresis Supervisor :Dr assadi By:A Pajand Birjandi ©2000 Timothy G. Standish Electrophoresis   Electro = flow of electricity, phoresis, from the Greek = to carry across A separation technique based on a solute’s ability to move through a conductive medium under the influence of an electric field. The medium is usually a buffered aqueous solution In the absence of other effects, cations migrate toward the cathode, and anions migrate toward the anode. More highly charged ions and ions of smaller size, which means they have a higher charge-to-size ratio, migrate at a faster rate ©2000 Timothy G. Standish    Factors Affecting Ionic Migration  The rate of migration reaches a constant value when the attractive force exerted by the electrode is balanced by the frictional force due to movement of the species through the electrolyte solution.  Each species is characterized by its electrophoretic mobility, µ,  total charge,  Its overall size  Shape  viscosity of the electrolyte solution.  The distance d travelled by a given species is related to its mobility µ and to the time of application and size of the potential gradient by the equation d=µt( E/S ) between the electrodes. d1- d2=(µ1-µ2)t(E/S) applied potential ©2000 Timothy G. Standish Effective factors on mobility:  pH  Temperature  Ionic Strength configurations include supporting media:  • Gels  • Packing  • Paper  • Capillaries ©2000 Timothy G. Standish ©2000 Timothy G. Standish Paper electrophoresis  The paper is saturated with a buffering solution and a sample introduced at one point  A dc potential of 100-1000 is applied  Species will migrate on the paper  After a period of time,the paper is removed and dried  If required the paper is treated with a color so that the bond can be observed ©2000 Timothy G. Standish ©2000 Timothy G. Standish What is Gel Electrophoresis?  A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin  Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied  Charged particles can include DNA, amino acids, peptides, etc ©2000 Timothy G. Standish Why do gel electrophoresis?  When DNA is cut by restriction enzymes, the result is a mix of pieces of DNA of different lengths  It is useful to be able to separate the pieces - I.e. for recovering particular pieces of DNA, for forensic work or for sequencing ©2000 Timothy G. Standish What is needed?   Agarose - a polysaccharide made from seaweed. Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with a network of crosslinked molecules Some gels are made with acrylamide if sharper bands are required ©2000 Timothy G. Standish    Buffer - in this case TBE The buffer provides ions in solution to ensure electrical conductivity. Not only is the agarose dissolved in buffer, but the gel slab is submerged (submarine gel) in buffer after hardening ©2000 Timothy G. Standish   Also needed are a power supply and a gel chamber Gel chambers come in a variety of models, from commercial through home-made, and a variety of sizes ©2000 Timothy G. Standish A gel being run Positive electrode Comb Agarose block DNA loaded in wells in the agaros Buffer Black background To make loading wells easier ©2000 Timothy G. Standish Steps in running a gel     DNA is prepared by digestion with restriction enzymes Agarose is made to an appropriate thickness (the higher the % agarose, the slower the big fragments run) and ‘melted’ in the microwave The gel chamber is set up, the ‘comb’ is inserted The agarose may have a DNA ‘dye’ added (or it may be stained later). The agarose is poured onto the gel block and cooled ©2000 Timothy G. Standish Next? The power source is turned on and the gel is run. The time of the run depends upon the amount of current and % gel, and requires experimentation  At the end of the run the gel is removed (it is actually quite stiff)  The gel is then visualized - UV light causes the bands of DNA to fluoresce  ©2000 Timothy G. Standish A gel as seen under UV light - some samples had 2 fragments of DNA, while others had none or one ©2000 Timothy G. Standish Submarine Gel Electrophoresis Sample to be loaded Negative electrode Gel Box Positive electrode Well Gel ©2000 Timothy G. Standish Submarine Gel Electrophoresis ©2000 Timothy G. Standish Submarine Gel Electrophoresis 100 V DNA Migration + ©2000 Timothy G. Standish Gel Electrophoresis Wells ©2000 Timothy G. Standish Gel Electrophoresis Wells ©2000 Timothy G. Standish Gel Electrophoresis Wells Large Direction of DNA Travel Small + ©2000 Timothy G. Standish Electrophoresis of Dyes 1 2 3 4 5 ©2000 Timothy G. Standish Positive Positive Positive Positive Positive _ Eco R I 21,226 bp 7421 bp 5804 and 5643 bp 4878 bp 3530 bp + ©2000 Timothy G. Standish Slab Gel Electrophoresis (SGE) ©2000 Timothy G. Standish Capillary Electrophoresis ©2000 Timothy G. Standish ©2000 Timothy G. Standish Characteristics -1 Electrophoresis in narrow-bore(25-150 μm id), fused silica capillaries High voltages (10-30 kV) and high electric fields applied across the capillary High resistance of the capillary limits current generation and internal heating High efficiency (N>105-106) Short analysis time(5-20 min) Detection performed on-capillary (no external detection cell) ©2000 Timothy G. Standish Characteristics -2 Small sample volume required (1-50 nlinjected) Limited quantities of chemicals and reagents required (financial and environmental benifits) Operates in aqueous media Simple instrumentation and method development Automated instrumentation Numerous modes to vary selectivity and wide application range Applicable to wider selection of analytes compared to other techniques (LC, TLC, SFC, cGC) Applicable to macro-and micromolecules Applicable to charged and neutral solutes Modern detector technology used (DAD, MS) ©2000 Timothy G. Standish Capillary Electrophoresis Apparatus ©2000 Timothy G. Standish Instrumentation Capillary electrophoresis: Power supply Anode compartment Both with buffer reservoir Cathod compartment narrow-bore fused-silica capillary tube; injection system; detector; Recorder ©2000 Timothy G. Standish ©2000 Timothy G. Standish Basics cont.   A photocathode is then used to measure the absorbencies of the molecules as they pass through the solution The absorbencies are analyzed by a computer and they are represented graphically ©2000 Timothy G. Standish Capillary electrophoresis   In capillary electrophoresis the sample is injected into a buffered solution retained within a capillary tube. components migrate as the result of two types of mobility: Electrophoretic mobility: A measure of a solute’s ability to move through a conductive medium in response to an applied electric field (mep). electroosmotic mobility: The movement of the conductive medium in response to an applied electric field. ©2000 Timothy G. Standish Equipment  Capillary tube Varied length but normally 25-50 cm  Small bore and thickness of the silica play a role – Using a smaller internal diameter and thicker walls help prevent Joule Heating, heating due to voltage  ©2000 Timothy G. Standish ©2000 Timothy G. Standish Equipment  Because ions are in the bulk solution are about to travel through the capillary without interference from the capillary itself, there is no dramatic drop in potential within the capillary  No meniscus is mad ©2000 Timothy G. Standish ©2000 Timothy G. Standish Parameters that effect EOF          • Electric field • Buffer pH • Ionic strength • Temperature • Organic modifier • Surfactants • Neutral hydrophilic polymer • Covalent coatings • External electric field ©2000 Timothy G. Standish ©2000 Timothy G. Standish Injection: • Pressure • Vacuum • Siphoning • Electrokinetic ©2000 Timothy G. Standish ©2000 Timothy G. Standish  Detector – – – – UV/Visible absorption Fluorescence Radiometric (for radioactive substances) Mass Spec. ©2000 Timothy G. Standish ©2000 Timothy G. Standish • CZE - capillary zone electrophoresis • CGE - capillary gel electrophoresis • CIEF - capillary isoelectric focusing • CITP - capillary isotachophoresis • CMEC - capillary micellar electrokinetic chromatography ©2000 Timothy G. Standish Modes of Operation ���� Capillary Zone Electrophoresis (CZE) ���� Capillary Isoelectric Focusing (CIEF) ���� Capillary Isotachophoresis (CITP) ���� Capillary Gel Electrophoresis (CGE) ���� Micellar Electrokinetic Chromatography (MEKC) ���� Microemulsion Electrokinetic Chromatography (MEEKC) ���� Non-Aqueous Capillary Electrophoresis ©2000 Timothy G. Standish        Sample Injection Port Light from UV source Sample (Analyte) UV detector Buffer (fixed pH) + -- CAPILLARY ZONE ELECTROPHORESIS ©2000 Timothy G. Standish Capillary zone electrophoresis Separation due to differences in charge, shape and size. ©2000 Timothy G. Standish Capillary gel electrophoresis Separation mainly due to differences in shape and size. ©2000 Timothy G. Standish Capillary isoelectric focusing Separation due to differences in isoelectric point (pI). ©2000 Timothy G. Standish Capillary isotachophoresis  Separation (sorting) due to mobility. ©2000 Timothy G. Standish Micellar electrokinetic chromatography Separation due to difference in hydrophobicity. ©2000 Timothy G. Standish ©2000 Timothy G. Standish ©2000 Timothy G. Standish What is the Lab-on-a-Chip? The Lab-on-a-chip is a generic term for a system or device designed to perform one or more chemical/biochemical process(es) leading to a controlled reaction or analysis. Also known as: • Micro Total Analysis Systems (μTAS) • Microfluidic systems First example: In 1979, miniaturised gas chromatograph fabricated in silicon at Stanford University (S.C. Terry) using standard microfabrication techniques from the micro-electronics industry. ©2000 Timothy G. Standish What is the Lab-on-a-Chip?  The Lab-on-a-Chip has rapidly developed as a field of research and technological development since the introduction of the concept of μTAS Traditionally, chemical analysis and medical diagnosis involves huge laboratories (usually centralised and remote), expensive specialised equipment, teams of skilled personnel and so on. Analysis or diagnosis involves a fluid based sample: • Medical and chemical analysis: typically water based • Environmental analysis: water, air A Lab-on-a-chip device therefore is based on the requirement to handle fluids, typically on very small scales: microfluidics. ©2000 Timothy G. Standish    Operations in microfluidic devices Typical processes performed during analysis: • Sample preparation (filtration, preconcentrating, purification…) • Reaction with chemical or molecular reagents • Separation (by mass, charge, size …) • Analyte or product detection or measurement Optical absorption – Fluorescence – Electrical – Mass spectrometry – Magnetic • Data collection and analysis ©2000 Timothy G. Standish CE on microchips  Presently microfluidic systems for capillary electrophoresis (CE) are being used. CE on microchips is one of the most promising technologies in µ-TAS. The CE is amenable to miniaturization because it involves the movement of fluids in a microchannel by electroosmosis. This precludes the need for mechanical pumps and valves, which are difficult to miniaturize and integrate into microdevices. ©2000 Timothy G. Standish   Capillary array electrophoresis on a chip: mask pattern for the 96channel radial capillary array electrophoresis microplate. ©2000 Timothy G. Standish  a microfluidic electrophoretic chip consists of an injection channel connecting the sample reservoir and the sample waste reservoir. The separation channel connects the buffer reservoir and the buffer waste reservoir. Capillary electrophoresis on a chip. (a) Schematic of the microchip used for PCR amplification and electrophoresis. The direction of arrows indicate injection (I) and separation (S). ©2000 Timothy G. Standish  In the injection mode, a fieldis applied between the sample reservoir and the sample waste, thus causing the DNA to migrate to the intersection cross. The small DNA plug at the intersection cross serves as an injection to the separation channel. In the separation mode, a field is applied between the buffer and the buffer waste reservoir. The moving buffer elutes the DNA mixture, which separates as it migrates down the separation channel. ©2000 Timothy G. Standish Detection methods – electrical Conductivity detection, Amperometric detection, ISFETs, Impedance analysis,Enzyme and label-free techniques. Detection methods - optical Fluorescence, absorption, refractive index, IR, integrated optics Comparison of separation of amino acids using HPLC (4 mm column), conventional capillary electrophoresis (CE) on 75 µm capillary, and chip technology (5×12 µm channel) ©2000 Timothy G. Standish ©2000 Timothy G. Standish