Presentazione sul tema: "ELETTROFORESI CON ELETTROLITA LIBERO Problemi di convezione"— Transcript della presentazione:
1 ELETTROFORESI CON ELETTROLITA LIBERO Problemi di convezione BASI DI ELETTROFORESIIl campo di forze che causa il trasporto differenziale e' di tipo elettrico.L'elettroforesi ha avuto il suo ideale campo di applicazione in bioanalitica.La separazione si realizza in base al rapporto carica/raggio della molecola.ELETTROFORESI CON ELETTROLITA LIBEROProblemi di convezioneELETTROFORESI CAPILLARE ELETTROFORESI CON SUPPORTO
2 Electrophoretic mobility Voltage difference, E = voltage applied/distance between electrodes; generally expressed as volts/cmCharge on molecule, qFrictional component, f, determined by size and shape of molecule, pore size of matrix, viscosity of bufferVelocity of particle, v= Eq/fMobility of particle, µ = v/E = q/fSize/shapeChargeBoth size/shape and chargeSeparation canbe effected by
3 Electrophoretic migration V = IR Ohm lawVoltage is a function of current and resistanceResistance decreases during electrophoretic run, therefore current increases if maintaining constant voltageWhy minimize current increase during run?As current increases, power increases- much of power is dissipated as heatHeat affects electrophoretic separation- diffusion increases; samples can be sensitive to heat; buffer viscosity decreases therefore resistance decreases and uneven heating occurs due to best cooling at gel edges
4 EFFICIENZA IN ELETTROFORESI Scan lucidoDT= coeff. diff. totaleq±1 in caso di altri meccanismi di dispersione
6 F z E = - = D m z EX V F N zV = 20 N z V T = F R 2 J EFFICIENZA IN ELETTROFORESIIl campo forza elettrica per mole di molecole sara' dato daFzE=-=DmextzEXVFV= caduta potenzialeSostituendo si ottiene che:NzVT=FR2JNUMERO DI PIATTIIn condizioni ideali si ottiene che:NzV=20Poiche'FCmol=96000/,V=100÷50000V,z=1÷10 si hache6N=2000÷10x10ALTA EFFICIENZA
8 ELETTROFORESI SU SUPPORTO Aumento del rapporto superficie/volumeTipi di supporto:acetato di cellulosacartagel di poliacrilammide (PAGE)agarosioI supporti danno un effetto di setaccio per separare in base alle dimensioni, uno volta che le specie siano state caricate in ambiente tamponatoEsempi di applicazioni:Analisi di proteine (Progetto “Proteoma”)Sequenziatura del DNA (Progetto “Genoma”)
9 DNA/Agarose Gel Electrophoresis Horizontal electrophoresis most common; simplest separation by sizeAgarose concentration 0.3-3%Buffer most often Tris-Borate-EDTA (TBE) at 1X or 0.5X; sometimes Tris-Acetate-EDTA (TAE) at 1X (Maniatis, Current Protocols)Detection of DNA is generally by ethidium bromide intercalation (dye in gel, in buffer, in sample, or in immersion solution after run) or by other dyes (e.g., Sypro)Agarose solution gels due to formation of inter- and intra-chain H bonds=> The higher the concentration, the smaller the pore size
10 DNA conformation and delectrophoretic mobility Plasmids, as well as viral and bacterial chromosomes, are circular molecules if untreated with nucleasesClosed circular DNA molecules can exist in several states, from closed circular to nicked to supercoiled to various levelsEach form migrates differently from the other and from linear DNA of the same size- supercoiled DNA has highest mobility and nicked closed circular has the lowestEthidium bromide, due to intercalation into DNA, affects supercoiling (intercalation decreases negative supercoiling), and can be used to determine extent of supercoiling
11 RNA/agarose gel electrophoresis Denaturation is critical. Formaldehyde gels and formamide-containing sample buffer are commonly used.Gels are cast in MOPS buffer with formaldehydeSamples are heated in formamide sample bufferGel must be run rapidly or buffer must be changed during longer runs- MOPS is not a strong bufferDetection of total RNA is often by EtBr staining- shows only major species (rRNAs)Detection of specific species most often is by northern blotting- transfer to nitrocellulose or other blotting paper, followed by hybridization with specific probes
12 ANALISI DEL DNA IN CHIMICA FORENSE Il DNA del sangue sugli indumenti dell’imputato è sovrapponibile a quello della vittima?…..Abby, la più popolare esperta di analisi del DNA per scopi investigativi (da NCIS, P. Perette)l, TS = controlliD = imputatojeans = macchie pantaloni imputatoshirt = macchie su maglietta imputatoV = DNA della vittimaLa probabilità di coincidenza casuale e’ di 1: 33x109
13 Detection of DNA on Southern blot Southern blotting is followed by hybridizing labeled DNA sequences to DNA immobilized on membrane, then by detection of labelRadioactivity by autoradiographyEnzyme by reaction to produce colored or luminescent productLabeled proteins to detect DNA binding
14 Southern blotting Developed by E. M. Southern Separated DNAs are transferred after electrophoresis to nitrocellulose or charged nylon membraneTransfer is by capillary action (below) or, less often, electrophoreticWeightPaper towelsFilter paperMembraneGelSupportFilter paper WickBuffer
15 Protein electrophoresis Proteins are sequence of amino acids that can be ionized depend on their acid or basic character. The N- and C- terminal and T-groups of the polypeptide can be ionized, contributing to the overall charge. The protein’s net electric charge is the sum of the electric charges found on the surface of the molecule as a function of the environment.- At the pI of a specific protein, the protein molecule carries no net charge and does not migrate in an electric field. - At pH above the pI, the protein has a net negative charge and migrates towards the anode. - At pH below the pI, the protein obtains a net positive charge on its surface and migrates towards the cathode.
16 Polyacrylamide Gel Electrophoresis (PAGE) Proteins, although can be used for separation of small DNAsVertical electrophoresis setup with thin gels (0.2-2 mm)Can be analytical or preparative scaleCan be denaturing (addition of SDS and reducing agent to sample and SDS to buffer; often also add denaturant to sample buffer; samples are heated before electrophoresis to ensure denaturation) or native conditionsSeparation: by size- denaturing; SDS treatment results in uniform charge densityby charge and size/shape- nativeby charge/pI- isoelectric focusing
17 PAGETotal percentage of acrylamide- acrylamide and bis-acrylamide- determines pore size of gelDiscontinuous gels are most common for highest resolution:Low percentage (3%) and low pH (6.8) are used for stacking gel- all proteins run readily through until hit higher percentage and pH (8.6) of running or separating gel (4-20%), then stack up due to change in pH.
18 Formazione di un gel PAIl setaccio tridimensionale si forma dalla co-polimerizzazione del monomero attivato (acrilammide) e del composto che forma i legami trasversali (metilen-bis-acrilammide)
19 Determinazione del MW via SDS PAGE La mobilità elettroforetica delle proteine in un gel SDS PAGE è inversamente proporzionale al logaritmo del loro peso molecolare
20 SDS-PAGE: MW separation 1. Denaturing method relying on two components: SDS and reducing agents2. Reducing agent ensures all disulfide bonds are reduced and SDS denatures and coats protein with basically uniform charge density3. Native charge masked and native shape lost so separation primarily by size. Linear relationship of logMW and Mr allows MW estimation from comparison with standard curve4. Separation may be quite different from gel to gel: protein standards should be included in each electrophoresis run. MW standards are also available to allow accurate MW determination of the proteins.
21 StainingSeparated proteins would not be visualized unless a dye (a stain) is used to give the protein color.Coomassie Blue - the fastest and the most commonly used stainZinc Stain - negatively stained protein on an opaque white background - ready in 10 minutesSYPRO Orange Stain - a fluorescent reagent - stained proteins are visualized by UV illuminationSilver Stain - highest sensitivity, 2 ng/band - ready in 1 hour
22 Coomassie stainingCoomassie Brilliant Blue is most common stain for protein gels.Staining is carried out in methanol + acetic acid, which acts to fix proteins in gel.Destaining is required to reduce background- methanol/acetic acid.Coomassie binds to most proteins with similar affinity, but not all.Binding is based on mostly ionic interaction (basic amino acids with -SO3- on Coomassie) plus some hydrophobic interaction with Coomassie rings.Lower limit for protein band detection by Coomassie staining is ~ ng.
24 Sypro staining Simple protocol. No overstaining. 1-4000 dynamic range. Less protein to protein variationStains glycoproteins, lipoproteins and Ca2+ binding proteins and other difficult-to-stain proteinsDo not stain DNA/RNAMS compatibleExpensive
25 Protein staining methods for proteomics SensitivityFeaturesSYPRO Ruby1 ng1. MS compatible2. High sensitivity3. Need special image acquiring instrument.Silver stain by Merril1. High sensitivity2. Glycoprotein and other low abundance proteins can be detectedSilver stain by Gottlieb*Coomassie Blue G-25010 ng2. Easy to handleCoomassie Blue R-250ng2. Low cost
27 Focalizzazione isoelettrica Un gradiente di pH si forma nel gel prima di caricare il campione.Caricato il campione, viene applicato il voltaggio. Le proteine migreranno fino al punto in cui il pH è uguale al loro pI, dove la loro carica netta è nulla.Le proteine formano bande che possono essere tagliate e usate per ulteriori esperimenti.
28 2D PAGE in proteomicaUn campione proteico è inizialmente frazionato nella prima dimensione mediante focalizzazione isoelettrica. Il gel di focalizzazione è quindi combinato con un PAGE in direzione ortogonale alla prima. Le proteine aventi stesso pI sono quindi separate in base al MW2D PAGE del proteoma di E.coli: si tratta di più di 1000 proteine
29 Analisi delle proteine plasmatiche Intervalli di normalità delle classi di proteine plasmaticheClasseIntervallo %Valori medi (g/dL)Albumine45-704.2Alfa(1)-globuline2-50.2Alfa(2)-globuline8-140.8Beta-globuline10-150.9Gamma-globuline11-221.2
30 Western blottingLe proteine vengono trasferite dal gel su un foglio di polimero (vedi Southern o Northern blotting) e marcate con anticorpo radioattivo, con sistemi avidina/biotina o traccianti colorati/luminescenti (tipo ELISA). In tal modo si rileva solo la proteina di interesse.Applicazione clinica tipica: test per epatite C
31 Capillary Zone Electrophoresis (CZE) Major drawbacks of gel electrophoresis: speed of analysis. Speed could be improved by increasing the electric current of the system.Large amount of heat would be generated: high convectionCZE uses silica fused capillaries ranging from to millimeters in outer diameter to dissipate the heat produced. Increasing the electric fields produces very efficient separations and reduces separation times.Very small amount of sample (0.1 to 10 nL) is required. The sample solution is injected at one end and a electric field of 100 to 700 volts/centimeter is applied across the capillary.
32 Electrophoresis in a buffer filled, narrow-bore capillaries CZE– The BasicsElectrophoresis in a buffer filled, narrow-bore capillariesEach capillary is about μm in internal diameterWhen a voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite chargeDepending on the charge, the molecules move through at different speedsSeparation is achieved
33 CZE– The Basics / IIA photocathode is then used to measure the absorbencies of the molecules as they pass through the solutionThe absorbencies are analyzed by a computer and they are represented graphically
34 CZE– The Basics/IIIThe movement of ions solely due to the electric field, potential differenceCations should migrate toward cathodeAnions should migrate toward anodeNeutral molecules do not favor either