Transcript Document

2D Electrophoresis:
1st Dimension
April 9, 2016
Proteomics Experimental Workflow
Isoelectric focusing - IPG rehydration
Focusing tray
IPGs are supplied as dry strips - on a
flexible plastic support
They must be rehydrated to at least their
original volume (0.5 mm thick x 3.3 mm
wide)
Rehydration tray
Rehydration can be done in a focusing
tray or a disposable rehydration tray.
First dimension – what is the isoelectric point (pI)?
•Proteins
•The
+
+
+
are amphoteric
charges are determined by
+
• amino acid composition
• pH of the environment
•Isoelectric
point
•IEF
is electrophoresis of proteins
in a pH gradient.
• Charged proteins move in an electric
field until they reach the point in the pH
gradient where their net charge is neutral.
pH < pI
pH = pI
pH > pI
First dimension – what is isoelectric focusing (IEF)?
9 5
3 6
Anode
+
pH
3
8
4
4
3 7
6
8
5
4 7
10
6
5
9
10
7
5 3
4
6 5
9 6
3
8
7
10
8
9
4
Cathode
9
-
10
Focusing with voltage
Anode
+
pH
3
3
3
3
44
4
4
3
4
5
5
55
5
66
6
6
6
77
7
8
8
8
7
8
99
9
9
9
10
10
10
Cathode
-
10
Proteins move in a pH gradient until they reach their isoelectric point (pI)
How do you run IEF?
Separation Medium:
IPG Strips
Instrument:
IEF Cell
IPG Strips
•IPG stands for immobilized pH gradient
•Acrylamide gels are poured with a pH gradient onto a
plastic backing, cut into strips and dehydrated
•pH gradients are created with sets of acrylamido buffers
which are derivatives of acrylamide containing both
reactive double bonds and buffering groups
•The pH gradient is fixed in the gel and doesn’t change
during focusing
•IPG strips allow for a high degree of reproducibility and
easy handling
IPG strips
IPG strips are available in a variety of pH gradients and lengths
Broad range
ReadyStrip™ IPG strips
Lengths
7 cm
11 cm
17 cm
18 cm
24 cm
pH 3-10
pH 3-10 nonlinear (NL)
Narrow Range
pH 3-6
pH 4-7
pH 5-8
pH 7-10
Micro Range
pH 3.9-5.1
pH 4.7-5.9
pH 5.5-6.7
pH 6.3-8.3
Broad Range pI Separations
pH 3-10
pH 3-10NL
IPG Strip Selection
pH 3 - 10
pH 3-10
pH 3 - 6
pH 3-6
pH 5 - 8
pH 5-8
pH 7 - 10
pH 7-10
E. coli Lysate
IPG Strip Resolution
Broad
3-10
Narrow
5-8
Micro
4.7-5.9
Sample/Rehydration buffer for IEF
ReadyPrep Rehydration/Sample Buffer
8 M urea,
2% CHAPS,
50 mM dithiothreitol (DTT),
0.2% (w/v) Bio-Lyte® 3/10 ampholytes,
Bromophenol Blue
问题：
1 加入这些目的是什么？
2 可以有替代品吗？ / 为什么不加其他东西？（PCR反应体系）
Laemmli Sample Buffer for 1D SDS-PAGE
Why do you use SDS in 1D
SDS-PAGE buffer and not
in rehydration/sample
buffer?
Component
Purpose
Tris HCl
Buffering Agent
SDS
Detergent: Disrupts hydrophobic interactions
and gives the proteinWhy
a net negative
do youcharge
use
Glycerol
βME or DTT
glycerol in 1D SDSGive the sample weight
PAGE buffer?
Reducing Agent
Why do you use DTT
in both 1D SDSPAGE buffer and
rehydration sample
buffer?
Protein Solubility
•加了一大堆东西，那么在什么情况下蛋白最容易溶解?
Separate polypeptide chains
•如何能够达到这个目的呢？
Breaking Molecular Interactions
•这些相互作用都包括什么呢？
covalent interactions - disulfide bridges
non-covalent interactions - ionic bonds,
hydrogen bonds
hydrophobic interactions
Disruption of Disulfide Bridges
Reducing agent
1 Mercaptoethanol - 700mM
碱性端的离子化，会破坏pH梯度
2 Dithiothreitol (DTT) - 50mM
无离子化，但并非完美，较多的半胱氨酸二硫键难以被还原
3 Tributylphosphine (TBP) - 2mM
挥发性、毒性、难闻气味、需要有机溶剂溶解，没有文献报道效
果比DTT好
Disruption of Ionic Bond & Hydrogen Bond
Chaotrope
改变介电常数，氢键形成和极化（疏水键）几乎所有和蛋白质溶
解相关的参数。
1 Urea
最主要的功能是打破氢键，其次是离子键，而打破离子键是通过
改变节电常数和蛋白质变性。
2 Thiourea
很强的致蛋白质变性作用，与Urea联合使用，明显促进蛋白质溶
解。
问题：可以单独使用吗？
Disruption of Hydrophobic Interactions
Detergent：
1 Ionic - SDS
完全不能用吗？
2 Nonionic - Triton, tween, NP-40, Brij, Mega
能用吗？
3 Zwitterionic - CHAPS, ASB
能用吗？
由于Urea的原因，在高浓度Urea情况下，CHAPS, Triton, NP-40
可以使用。
Sample/Rehydration buffer for IEF
Component
Purpose
Urea
Chaotropic agent: Disrupts hydrogen bonds
and prevents aggregation and formation of 2º
structures, helps solublization
Reducing agents: Disrupts disulphide bonds
so proteins remain as single subunits
DTT
CHAPS
Detergent: Disrupts hydrophobic interactions
Carrier Ampholytes
Help counteract insufficient salt in a sample
Bromophenol Blue
Allows monitoring of the run
Isoelectric focusing – rehydration & sample loading
Passive Rehydration: no voltage, sample is in rehydration buffer
Active Rehydration: low voltage, sample is in rehydration buffer
Cup Loading: rehydrate with buffer, add sample to cup with low voltage
Pros and cons of different rehydration techniques
Technique
Advantages
Disadvantages
Passive
Rehydration
•Sample
•Large
•Avoids
application is simple
the problem of sample precipitation
proteins may not enter the
strip
•Shorter
focusing times can be used because the proteins are in
the strip before focusing step
•Large
amounts of proteins can be loaded
•This
allows the advantage of rehydrating in a separate tray
while using the cell to focus another batch of strips
Active
Rehydration
•Sample
application is simple
•Proteins
•Avoids
enter gel by absorption and electrical pull
•small
proteins with a higher
mobility have a higher risk of
being lost from the strip
the problem of sample precipitation
•Shorter
focusing times can be used because the proteins are in
the strip before focusing step
•Large
Cup Loading
amounts of proteins can be loaded
Good for samples that contain high levels of DNA, RNA or other
large molecules
•Sample
precipitation can occur
For serum samples that have not been treated to remove
albumin
complicated, cup must be
sealed to the surface of the prerehydrated empty strip
When running basic IPG strips (e.g. pH 7-10)
•IPG
For samples that contain high concentrations of glycoproteins
•More
strip must be rehydrated
prior to sample application
Power conditions/programming
•Strips should always be run at the highest voltage
compatible with the heat dissipation capacities of the cell
Example Protocol
•Volt hours: time integral of applied voltage. A standard
for reproducing focusing conditions. This needs to be
determined empirically
Step
1
Step
2
250V for 1 h.
Step
3
8,000 V for 26,000
Volt-hours
Step
4
Hold at 750 V
•When an electrical field is applied to an IPG strip at the
beginning of a run, the current will be high because of
the high number of charged carriers present, as the
proteins and ampholytes migrate, the current will
gradually decrease because of decreasing charge
•Similar strips and samples should be run in batches so
that the electrical conditions will be as consistent as
possible
•Voltage is limited by current. If the maximum current is
reached before the maximum voltage, the voltage will
not reach the maximum
Slow ramp to
8,000V over 1 h