FACOLTA DI SCIENZE FF MM NN – TECNICHE ANALITICHE SEPARATIVE– LM Chimica Il campo di forze che...
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FACOLTA’ DI SCIENZE FF MM NN – TECNICHE ANALITICHE SEPARATIVE– LM Chimica 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 BASI DI ELETTROFORESI
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Transcript of FACOLTA DI SCIENZE FF MM NN – TECNICHE ANALITICHE SEPARATIVE– LM Chimica Il campo di forze che...
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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
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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
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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
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Scan lucido
DT= coeff. diff. totale±1 in caso di altri meccanismi di dispersione
EFFICIENZA IN ELETTROFORESI
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Scan lucido
EFFICIENZA IN ELETTROFORESI
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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
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ELETTROFORESI SU SUPPORTO
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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
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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
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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
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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
o Detection of total RNA is often by EtBr staining- shows only major species (rRNAs)
o Detection of specific species most often is by northern blotting- transfer to nitrocellulose or other blotting paper, followed by hybridization with specific probes
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ANALISI DEL DNA IN CHIMICA FORENSE
…..Abby, la più popolare esperta di analisi del DNA per scopi investigativi (da NCIS, P. Perette)
, TS = controlli
D = imputato
jeans = macchie pantaloni imputato
shirt = macchie su maglietta imputato
V = DNA della vittima
Il DNA del sangue sugli indumenti dell’imputato è sovrapponibile a quello della vittima?
La probabilità di coincidenza casuale e’ di 1: 33x109
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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
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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
Filter paper
Gel
Support
Buffer
Membrane
Filter paper Wick
Paper towels
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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
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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
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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.
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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)
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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
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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.
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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
Staining
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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.
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Covalent/Non-covalent silver staining
1. Covalent silver staining (with glutardialdehyde)
1. More sensitive
2. MS incompatible
3. Lower background
4. No negative staining
2. Non- Covalent silver staining (without glutardialdehyde)
1. Less sensitive
2. MS compatible
3. Higher background
4. Some proteins negative stained
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Sypro staining
1. Simple protocol. No overstaining.
2. 1-4000 dynamic range.
3. Less protein to protein variation
4. Stains glycoproteins, lipoproteins and Ca2+ binding proteins and other difficult-to-stain proteins
5. Do not stain DNA/RNA
6. MS compatible
7. Expensive
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Protein staining methods for proteomics
Method Sensitivity Features
SYPRO Ruby 1 ng 1. MS compatible2. High sensitivity3. Need special image acquiring
instrument.
Silver stain by Merril
1 ng 1. High sensitivity2. Glycoprotein and other low
abundance proteins can be detected
Silver stain by Gottlieb*
1 ng 1. MS compatible2. High sensitivity
Coomassie Blue G-250
10 ng 1. MS compatible2. Easy to handle
Coomassie Blue R-250
50-100 ng 1. MS compatible2. Low cost
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Charge of a protein vs. pH
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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.
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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
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Analisi delle proteine plasmaticheProteina % proteine totali Vel. elettroforetica
pre-albumina <0.5 9.0
albumina 55.0 6.1
glicoproteine <0.5 5.1
5.0
inibitori tripsina
lipoproteine 3.0
glicoproteine 1.2 4.2
macroglobuline <4.5 4.2
mucoproteine 0.5 4.9
plasminogeno 4.7
aptoglobine
Fattore V 4.7
beta-globulina 3.0
transferrina <6.5 3.3
Fattore VIII 2.9
lipoproteine <14
fibrinogeno 2.1
beta(2)-globulina 3.0
b(2)A-globulina
gamma-globuline <17.2
isoemmaglutinine 1.1
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
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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
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•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)
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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
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a • 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
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a – 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
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CZE– Basic theory
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CZE– Basic theory/II
f
qep
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CZE– Basic theory/III
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CZE– Basic theory/IV
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CZE– Il flusso elettroosmotico
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CZE– Flusso elettroosmotico anodico
Se la parete del capillare viene caricata positivamente allora:
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CZE– Il flusso elettroosmotico/II
= 0
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CZE– Il Potenziale
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CZE– Migrazione
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CZE– Migrazione/II
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CZE– Migrazione/III
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
CZE– Efficienza
Nz V
T F
R2
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
CZE– Aspetti strumentali
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
CZE– Aspetti strumentali/II
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
CZE– Rivelatori
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
CZE– Rivelatori
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
CZE– UV/vis
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
CZE– UV/vis /II
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
CZE– MS
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– T
EC
NIC
HE
AN
AL
ITIC
HE
SE
PA
RA
TIV
E–
LM
Ch
imic
a
Seminario Prof. De LorenziUniversità di Pavia
CZE e tecniche ibride
