sedimentation, electrophoresis -...
TRANSCRIPT
Sedimentation Electrophoresis
Biophysics seminar Taliaacuten Csaba Gaacutebor Kollaacuter Veronika
27-28 03 2012
Why do we need to centrifuge
The sedimentation is 1 cm
The molecular weight of the protein 50 kDa = 50 kgmol
23
23
50kg molm 833 10 kg
6 10 mol
Temperature (T) 20 degC = 293 degK ∆h=1 cm= 001 m
The change of the Potential Energy
k-Boltzmann constant 138middot10minus23 JK
The vertical component of Kinetic Energy
kz
23 21
1E k T
2
J05 138 10 293K 202 10 J
K21
24
202 10 J250
817 10 J
23 24
pot 2
mE m g h 833 10 kg 981 001m 817 10 J
s
Density gradient centrifugation
2mrF lcentrifuga
2
buoyancy k
mF r
If ρ = ρk then Ff = mrω2 = Ff AND v = 0
High molecular weight and small size CsCl CsBr glycerol saccharose
Meselson-Stahl experiment (1958)
Matthew Meselson American biologist
(1930-)
Franklin Stahl American biologist
(1929-)
Not dispersive
14N
Not conservative
Example
rnracp
222 4
rNrN
acp
2
2
2 011060
4
RCF= 21000g a=21000x10=210000
r
aN
cp
0110
g
aRCF
cp
You would like to separate one protein from the cell debris therefore
you need to apply a centrifugal force of 21000 g The radius of the rotor
is 12 cm Which RPM would you use for UC to achieve the separation
n- revolutions per second N-RPM r-radius (m)
N=12613 rpm
Elecrophoresis
Movement of charged molecule in electrostatic field
Types of electrophoresis
I Free electrophoresis
II Gel electrophoresis
1 Agarose gel electrophoresis
2 Poliakrilamide gel electrophoresis
3 Gradient gel electrophoresis
4 Izoelectric focusing
5 Two dimensional electrophoresis
III Blotting techniques
1 Southern Blot
2 NorthernBlot
3 Western Blot
IV Capillary electrophoresis
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Why do we need to centrifuge
The sedimentation is 1 cm
The molecular weight of the protein 50 kDa = 50 kgmol
23
23
50kg molm 833 10 kg
6 10 mol
Temperature (T) 20 degC = 293 degK ∆h=1 cm= 001 m
The change of the Potential Energy
k-Boltzmann constant 138middot10minus23 JK
The vertical component of Kinetic Energy
kz
23 21
1E k T
2
J05 138 10 293K 202 10 J
K21
24
202 10 J250
817 10 J
23 24
pot 2
mE m g h 833 10 kg 981 001m 817 10 J
s
Density gradient centrifugation
2mrF lcentrifuga
2
buoyancy k
mF r
If ρ = ρk then Ff = mrω2 = Ff AND v = 0
High molecular weight and small size CsCl CsBr glycerol saccharose
Meselson-Stahl experiment (1958)
Matthew Meselson American biologist
(1930-)
Franklin Stahl American biologist
(1929-)
Not dispersive
14N
Not conservative
Example
rnracp
222 4
rNrN
acp
2
2
2 011060
4
RCF= 21000g a=21000x10=210000
r
aN
cp
0110
g
aRCF
cp
You would like to separate one protein from the cell debris therefore
you need to apply a centrifugal force of 21000 g The radius of the rotor
is 12 cm Which RPM would you use for UC to achieve the separation
n- revolutions per second N-RPM r-radius (m)
N=12613 rpm
Elecrophoresis
Movement of charged molecule in electrostatic field
Types of electrophoresis
I Free electrophoresis
II Gel electrophoresis
1 Agarose gel electrophoresis
2 Poliakrilamide gel electrophoresis
3 Gradient gel electrophoresis
4 Izoelectric focusing
5 Two dimensional electrophoresis
III Blotting techniques
1 Southern Blot
2 NorthernBlot
3 Western Blot
IV Capillary electrophoresis
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Density gradient centrifugation
2mrF lcentrifuga
2
buoyancy k
mF r
If ρ = ρk then Ff = mrω2 = Ff AND v = 0
High molecular weight and small size CsCl CsBr glycerol saccharose
Meselson-Stahl experiment (1958)
Matthew Meselson American biologist
(1930-)
Franklin Stahl American biologist
(1929-)
Not dispersive
14N
Not conservative
Example
rnracp
222 4
rNrN
acp
2
2
2 011060
4
RCF= 21000g a=21000x10=210000
r
aN
cp
0110
g
aRCF
cp
You would like to separate one protein from the cell debris therefore
you need to apply a centrifugal force of 21000 g The radius of the rotor
is 12 cm Which RPM would you use for UC to achieve the separation
n- revolutions per second N-RPM r-radius (m)
N=12613 rpm
Elecrophoresis
Movement of charged molecule in electrostatic field
Types of electrophoresis
I Free electrophoresis
II Gel electrophoresis
1 Agarose gel electrophoresis
2 Poliakrilamide gel electrophoresis
3 Gradient gel electrophoresis
4 Izoelectric focusing
5 Two dimensional electrophoresis
III Blotting techniques
1 Southern Blot
2 NorthernBlot
3 Western Blot
IV Capillary electrophoresis
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Meselson-Stahl experiment (1958)
Matthew Meselson American biologist
(1930-)
Franklin Stahl American biologist
(1929-)
Not dispersive
14N
Not conservative
Example
rnracp
222 4
rNrN
acp
2
2
2 011060
4
RCF= 21000g a=21000x10=210000
r
aN
cp
0110
g
aRCF
cp
You would like to separate one protein from the cell debris therefore
you need to apply a centrifugal force of 21000 g The radius of the rotor
is 12 cm Which RPM would you use for UC to achieve the separation
n- revolutions per second N-RPM r-radius (m)
N=12613 rpm
Elecrophoresis
Movement of charged molecule in electrostatic field
Types of electrophoresis
I Free electrophoresis
II Gel electrophoresis
1 Agarose gel electrophoresis
2 Poliakrilamide gel electrophoresis
3 Gradient gel electrophoresis
4 Izoelectric focusing
5 Two dimensional electrophoresis
III Blotting techniques
1 Southern Blot
2 NorthernBlot
3 Western Blot
IV Capillary electrophoresis
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Not dispersive
14N
Not conservative
Example
rnracp
222 4
rNrN
acp
2
2
2 011060
4
RCF= 21000g a=21000x10=210000
r
aN
cp
0110
g
aRCF
cp
You would like to separate one protein from the cell debris therefore
you need to apply a centrifugal force of 21000 g The radius of the rotor
is 12 cm Which RPM would you use for UC to achieve the separation
n- revolutions per second N-RPM r-radius (m)
N=12613 rpm
Elecrophoresis
Movement of charged molecule in electrostatic field
Types of electrophoresis
I Free electrophoresis
II Gel electrophoresis
1 Agarose gel electrophoresis
2 Poliakrilamide gel electrophoresis
3 Gradient gel electrophoresis
4 Izoelectric focusing
5 Two dimensional electrophoresis
III Blotting techniques
1 Southern Blot
2 NorthernBlot
3 Western Blot
IV Capillary electrophoresis
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Example
rnracp
222 4
rNrN
acp
2
2
2 011060
4
RCF= 21000g a=21000x10=210000
r
aN
cp
0110
g
aRCF
cp
You would like to separate one protein from the cell debris therefore
you need to apply a centrifugal force of 21000 g The radius of the rotor
is 12 cm Which RPM would you use for UC to achieve the separation
n- revolutions per second N-RPM r-radius (m)
N=12613 rpm
Elecrophoresis
Movement of charged molecule in electrostatic field
Types of electrophoresis
I Free electrophoresis
II Gel electrophoresis
1 Agarose gel electrophoresis
2 Poliakrilamide gel electrophoresis
3 Gradient gel electrophoresis
4 Izoelectric focusing
5 Two dimensional electrophoresis
III Blotting techniques
1 Southern Blot
2 NorthernBlot
3 Western Blot
IV Capillary electrophoresis
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Elecrophoresis
Movement of charged molecule in electrostatic field
Types of electrophoresis
I Free electrophoresis
II Gel electrophoresis
1 Agarose gel electrophoresis
2 Poliakrilamide gel electrophoresis
3 Gradient gel electrophoresis
4 Izoelectric focusing
5 Two dimensional electrophoresis
III Blotting techniques
1 Southern Blot
2 NorthernBlot
3 Western Blot
IV Capillary electrophoresis
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Types of electrophoresis
I Free electrophoresis
II Gel electrophoresis
1 Agarose gel electrophoresis
2 Poliakrilamide gel electrophoresis
3 Gradient gel electrophoresis
4 Izoelectric focusing
5 Two dimensional electrophoresis
III Blotting techniques
1 Southern Blot
2 NorthernBlot
3 Western Blot
IV Capillary electrophoresis
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
SDS Polyacrylamide gelelectrophoresis (SDS-PAGE)
(sodium-dodecyl-sulphate)
TEMED (Tetramethyl-ethylenediamine)
ammonium-persulphate
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Discontinuous PAGE
pH = 83
pH = 68 5 acrylamide
pH = 89 8-12 acrylamide
pH = 83
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Stacking gel pH = 68 neutral form of dominance Low electrophoretic mobility
Separating gel pH = 89 negative form of dominance high electrophoretic mobility
The mobility of the chloride ion is higher 1 Cl- drifts faster alone in the lower zone 2 The resitance of the zone is less Zones connected in series the current (I) is the same The voltage (V = I R) will be higher in the glycine zone
V
If a glycine molecule goes ahead because of lower voltage it will slow down If a Cl- lags behind because of higher voltage it will speed up The separation of the ion phase and the voltage gradient intensify each other
In the stacking gel Voltage gradient
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
In the stacking gel The mobility of the proteins is between that of the chloride and glycine so the proteins run between chloride and Gly in a narrow band
In the separating gel The glycine has negative charge at higher pH so it has also higher mobility The sequence of the mobility Cl- glycine proteins The higher pH value raises the mobility of the proteins The lower poresize (higher acrylamide ) reduces the mobility The separation is according to size
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Blotting techniques
Edwin Southern English biochemist
(1938-)
Southern specific DNA-sequence DNA-DNA hybridization Northern specific RNA-sequence RNA-DNA hybridization Western specific protein sequence protein-antigen Eastern post-syntetic protein modification phosphate carbohydrate lipid
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Southern Northern blot
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Western blot
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
buffer
buffer
electrode
capillary
sample
detector
electrode
High voltage
The differential movement for migration of ions by attraction
or repulsion in an electric field
Depending on the charge the molecules move through at
different speeds
Detection OD λ = 200 nm
bull small size big charge ndashbig
mobilityfast
bull big size small charge ndashsmall
mobilityslow
Capillary electrophoresis
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
The base of the method
The elecroosmotic flow (EOF)
The elecroosmotic flow formation
inner surface of negative charge quartz capillary (Si-O-)
hydrated cations accumulate near the surface
The electric field causes mass flow
Double layer in the wall of capillary
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions
Low heat development
Short time fast
Relatively inexpensive
Small sample (1-10 nl)
Automated
The advantages of this technique
Migration
1 Cations
2 Neutral molecules
3 Anions