ELETTROFORESI CON ELETTROLITA LIBERO Problemi di convezione BASI DI ELETTROFORESI   Il 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 LIBERO Problemi di convezione ELETTROFORESI CAPILLARE ELETTROFORESI CON SUPPORTO

Electrophoretic mobility Voltage difference, E = voltage applied/distance between electrodes; generally expressed as volts/cm Charge on molecule, q Frictional component, f, determined by size and shape of molecule, pore size of matrix, viscosity of buffer Velocity of particle, v= Eq/f Mobility of particle, µ = v/E = q/f Size/shape Charge Both size/shape and charge Separation can be effected by

Electrophoretic migration V = IR Ohm law Voltage is a function of current and resistance Resistance decreases during electrophoretic run, therefore current increases if maintaining constant voltage Why minimize current increase during run? As current increases, power increases- much of power is dissipated as heat Heat 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

EFFICIENZA IN ELETTROFORESI Scan lucido DT= coeff. diff. totale q±1 in caso di altri meccanismi di dispersione

EFFICIENZA IN ELETTROFORESI Scan lucido

F z E = - = D m z EX V F N zV = 20 N z V T = F R 2 J EFFICIENZA IN ELETTROFORESI Il campo forza elettrica per mole di molecole sara' dato da F z E = - = D m ext z EX V F V= caduta potenziale Sostituendo si ottiene che: N z V T = F R 2 J NUMERO DI PIATTI In condizioni ideali si ottiene che: N zV = 20 Poiche' F C mol = 96000 / , V= 100÷50000V, z =1÷10 si ha che 6 N=2000÷10x10 ALTA EFFICIENZA

ELETTROFORESI SU SUPPORTO

ELETTROFORESI SU SUPPORTO Aumento del rapporto superficie/volume   Tipi di supporto: acetato di cellulosa carta gel di poliacrilammide (PAGE) agarosio I supporti danno un effetto di setaccio per separare in base alle dimensioni, uno volta che le specie siano state caricate in ambiente tamponato Esempi di applicazioni: Analisi di proteine (Progetto “Proteoma”) Sequenziatura del DNA (Progetto “Genoma”)

DNA/Agarose Gel Electrophoresis Horizontal electrophoresis most common; simplest separation by size Agarose 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

DNA conformation and delectrophoretic mobility Plasmids, as well as viral and bacterial chromosomes, are circular molecules if untreated with nucleases Closed circular DNA molecules can exist in several states, from closed circular to nicked to supercoiled to various levels Each 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 lowest Ethidium bromide, due to intercalation into DNA, affects supercoiling (intercalation decreases negative supercoiling), and can be used to determine extent of supercoiling

RNA/agarose gel electrophoresis Denaturation is critical. Formaldehyde gels and formamide-containing sample buffer are commonly used. Gels are cast in MOPS buffer with formaldehyde Samples are heated in formamide sample buffer Gel must be run rapidly or buffer must be changed during longer runs- MOPS is not a strong buffer Detection 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

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 = controlli D = imputato jeans = macchie pantaloni imputato shirt = macchie su maglietta imputato V = DNA della vittima La probabilità di coincidenza casuale e’ di 1: 33x109

Detection of DNA on Southern blot Southern blotting is followed by hybridizing labeled DNA sequences to DNA immobilized on membrane, then by detection of label Radioactivity by autoradiography Enzyme by reaction to produce colored or luminescent product Labeled proteins to detect DNA binding

Southern blotting Developed by E. M. Southern Separated DNAs are transferred after electrophoresis to nitrocellulose or charged nylon membrane Transfer is by capillary action (below) or, less often, electrophoretic Weight Paper towels Filter paper Membrane Gel Support Filter paper Wick Buffer

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.

Polyacrylamide Gel Electrophoresis (PAGE) Proteins, although can be used for separation of small DNAs Vertical electrophoresis setup with thin gels (0.2-2 mm) Can be analytical or preparative scale Can 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 conditions Separation: by size- denaturing; SDS treatment results in uniform charge density by charge and size/shape- native by charge/pI- isoelectric focusing

PAGE Total percentage of acrylamide- acrylamide and bis-acrylamide- determines pore size of gel Discontinuous 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.

Formazione di un gel PA Il setaccio tridimensionale si forma dalla co-polimerizzazione del monomero attivato (acrilammide) e del composto che forma i legami trasversali (metilen-bis-acrilammide)

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

SDS-PAGE: MW separation 1. Denaturing method relying on two components: SDS and reducing agents 2. Reducing agent ensures all disulfide bonds are reduced and SDS denatures and coats protein with basically uniform charge density 3. 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 curve 4. 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.

Staining Separated 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 stain                        Zinc Stain     - negatively stained protein on an opaque        white background     - ready in 10 minutes                        SYPRO Orange Stain     - a fluorescent reagent     - stained proteins are visualized by UV         illumination                        Silver Stain     - highest sensitivity, 2 ng/band     - ready in 1 hour

Coomassie staining Coomassie 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 ~10-100 ng.

Covalent/Non-covalent silver staining Covalent silver staining (with glutardialdehyde) More sensitive MS incompatible Lower background No negative staining Non- Covalent silver staining (without glutardialdehyde) Less sensitive MS compatible Higher background Some proteins negative stained

Sypro staining Simple protocol. No overstaining. 1-4000 dynamic range. Less protein to protein variation Stains glycoproteins, lipoproteins and Ca2+ binding proteins and other difficult-to-stain proteins Do not stain DNA/RNA MS compatible Expensive

Protein staining methods for proteomics Sensitivity Features SYPRO Ruby 1 ng 1. MS compatible 2. High sensitivity 3. Need special image acquiring instrument. Silver stain by Merril 1. High sensitivity 2. Glycoprotein and other low abundance proteins can be detected Silver stain by Gottlieb* Coomassie Blue G-250 10 ng 2. Easy to handle Coomassie Blue R-250 50-100 ng 2. Low cost

Charge of a protein vs. pH

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.

2D PAGE in proteomica Un 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 MW 2D PAGE del proteoma di E.coli: si tratta di più di 1000 proteine

Analisi delle proteine plasmatiche Intervalli di normalità delle classi di proteine plasmatiche Classe Intervallo % Valori medi (g/dL) Albumine 45-70 4.2 Alfa(1)-globuline 2-5 0.2 Alfa(2)-globuline 8-14 0.8 Beta-globuline 10-15 0.9 Gamma-globuline 11-22 1.2

Western blotting Le 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

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 convection CZE uses silica fused capillaries ranging from 0.150 to 0.375 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.

Electrophoresis in a buffer filled, narrow-bore capillaries CZE– The Basics Electrophoresis in a buffer filled, narrow-bore capillaries Each capillary is about 25-100 μm in internal diameter When a voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite charge Depending on the charge, the molecules move through at different speeds Separation is achieved

CZE– The Basics / II 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

CZE– The Basics/III The movement of ions solely due to the electric field, potential difference Cations should migrate toward cathode Anions should migrate toward anode Neutral molecules do not favor either

CZE– Basic theory

CZE– Basic theory/II

CZE– Basic theory/III

CZE– Basic theory/IV

CZE– Il flusso elettroosmotico

CZE– Flusso elettroosmotico anodico Se la parete del capillare viene caricata positivamente allora:

CZE– Il flusso elettroosmotico/II = 0

CZE– Il Potenziale z

CZE– Migrazione

CZE– Migrazione/II

CZE– Migrazione/III

CZE– Efficienza N z V T = F R 2 J

CZE– Aspetti strumentali

CZE– Aspetti strumentali/II

CZE– Rivelatori

CZE– Rivelatori

CZE– UV/vis

CZE– UV/vis /II

CZE– MS

Seminario Prof. De Lorenzi CZE e tecniche ibride Seminario Prof. De Lorenzi Università di Pavia