Upload
francesco-de-rossi
View
219
Download
0
Embed Size (px)
Citation preview
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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 LIBEROProblemi di convezione
ELETTROFORESI CAPILLARE ELETTROFORESI CON SUPPORTO
BASI DI ELETTROFORESI
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
V = IR Ohm lawVoltage 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?
o As current increases, power increases- much of power is dissipated as heat
o 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
Electrophoretic migration
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
Scan lucido
DT= coeff. diff. totale±1 in caso di altri meccanismi di dispersione
EFFICIENZA IN ELETTROFORESI
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
Scan lucido
EFFICIENZA IN ELETTROFORESI
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
Il campo forza elettrica per mole di molecole sara' dato da
F z E F
ext z EX z VF F V= caduta potenziale
Sostituendo si ottiene che:
Nz V
T F
R2 NUMERO DI PIATTI
In condizioni ideali si ottiene che:
N zV20
Poiche' F C mol= 96000 / , V= 100÷50000V, z=1÷10 si ha che
N=2000÷10x106 ALTA EFFICIENZA
EFFICIENZA IN ELETTROFORESI
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
Aumento del rapporto superficie/volume Tipi di supporto:
acetato di cellulosacarta 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”)
ELETTROFORESI SU SUPPORTO
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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.
Protein electrophoresis
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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.
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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)
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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.
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
Charge of a protein vs. pH
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
Focalizzazione isoelettrica
Un gradiente di pH si forma nel gel prima di caricare il campione.
(A) 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.
(B) Le proteine formano bande che possono essere tagliate e usate per ulteriori esperimenti.
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
2D PAGE in proteomica
(A) 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
(B) 2D PAGE del proteoma di E.coli: si tratta di più di 1000 proteine
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
ELETTROFORESI SU SUPPORTO
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
•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.
Capillary Zone Electrophoresis (CZE)
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
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
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
• 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 / II
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
– 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– The Basics/III
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Basic theory
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Basic theory/II
f
qep
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Basic theory/III
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Basic theory/IV
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Il flusso elettroosmotico
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Flusso elettroosmotico anodico
Se la parete del capillare viene caricata positivamente allora:
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Il flusso elettroosmotico/II
= 0
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Il Potenziale
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Migrazione
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Migrazione/II
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Migrazione/III
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Rivelatori
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– Rivelatori
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– UV/vis
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
CZE– UV/vis /II
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
–T
EC
NIC
HE
SE
PA
RA
TIV
E –
LM
C
Seminario Prof. De LorenziUniversità di Pavia
CZE e tecniche ibride