2-DE, Isoelectric focusing

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Proteomics

• Session 7
• 2-DE, Isoelectric focusing

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First dimension, Isoelectric focusing (IEF)
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What is IEF

• IEF is preformed in a pH gradient.
• Proteins are amphoteric molecules with acidic and
  basic buffering groups (side chain).
• In basic environment, the acidic groups become
  negatively charged.
• In acidic environment, the basic groups become
  positively charged.
• The net charge of a protein is the sum of all
  charges.
• Isoelectric point (pI) the pH where the charge
  of a protein is zero.

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The principle of IEF
The IEF is a very high resolution separation
method, and the pI of a protein can be measured.
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Titration curve analysis
1. Forms pH 3-10 gradient first
2. Sample applied here (mixture of proteins)
More proteins have steeper curves below their pI.
This is why mostly sample application close to
the anode results in better patterns.
3. Electric field applied
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2-DE instruments, 1st dimension
Amersham Biosciences
Bio-Rad
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Two ways to form pH gradient
A. Classic IEF technique Carrier ampholyte
generated pH gradient. B. modern IEF technique
Immobilized pH gradients
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A. Carrier ampholyte generated pH gradient

• First developed by Svensson, nature pH gradient
  (1961).Svensson H. Acta Chem Scand (1961) vol.15,
  p325.
• Practical realization by Vesterberg (1969).
• Artificial pH gradient synthesis of a
  heterogeneous mixtures of isomers of aliphatic
  oligoamino-oligocarboxylic acid.

CH2
(CH2)x
N
(CH2)x
N
(CH2)x
(CH2)x
(CH2)x
R H or (CH2)x-COOH, X2 or 3
NR2
COOH
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Synthetic carrier ampholyte v.s. natural
occurring ampholyte

• Synthetic carrier ampholyte
• High buffering capacity and solubility at the pI.
• Good and regular electric conductivity at the pI.
• Absence of biological effects.
• Low molecular weight.
• Natural occurring ampholyte
• Amino acids or peptides
• Lack the properties above
• Can not be used in IEF.

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Behavior of ampholytes

• Negatively (-) charged carrier ampholyte
• Move toward anode ().
• Such as -COO-
• Positively () charged carrier ampholyte
• Move toward cathode (-).
• Such as NH3

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Solution in the IEF

• To maintain a gradient as stable as possible,
  electrode solutions are applied between the gel
  and the electrodes.
• Acid is used at the anode.
• Base is used at the cathode.
• Example an acidic carrier ampholyte reach the
  anode (), its basic buffering group would
  protonated (acquire a positive charge) from the
  medium and it would be attracted back by the
  cathode.

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Carrier ampholytes as solvents for proteins.

• Carrier ampholytes also help to solublize
  proteins, which stay in solution only in the
  presence of buffering compounds.
• They are necessary in traditional IEF and new
  immobilized pH gradient IEF.

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Problems for the traditional IEF

• 1. Long running time.
• Protein close to their pI have low net charge
  thus have low mobility.
• Denatured polypeptides migrate slower in gel than
  native protein.
• 2. Gradient drift.
• The pH gradient become instable during lone time
• Most basic proteins drift out of the gel.
• 3. Proteins behave like additional carrier
  ampholyte
• They modify the profile of pH gradient

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B. Immobilized pH gradient, IPG

• First developed by Righetti ,(1990).
• Immobilized pH gradient generated by buffering
  acrylamide derivatives (Immobilines)
• Immobilines are weak acid or weak base.
• General structure

N
N
CH2
C
CN
H
CH2
C
CN
R
H
O
H
O
R amino or carboxylic groups
Acrylamide
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Schematic drawing of a IPG
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Preparing an immobilized pH gradient

• Immobiline 0.2M
• Gel conc. 4T, 3 C
• Acidic solution is usually made denser by adding
  glycerol
• The gel is usually 0.5 mm thick

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Commercial immobilized pH gradient strips (IPG
strips)

• Introduced by Gorg. A.
• Ref Gorg. A (1994), Westermeier (2001)
• Dried gel strips can be stored at -20 to -80 from
  months to years.

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Advantage of IPG strips

1. Industrial standard (GMP) reduce variation.
2. The chemistry of the immobiline is better
   controllable.
3. The film-supported gel strips are easy to handle.
4. The fixed gradient are consistent during IEF.
5. Stable basic pH gradient allow reproducible
   results for basic proteins.
6. High protein loads are achievable.
7. Less protein loss during equilibration in SDS
   buffer.

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Gradient type (Amersham Bioscience)
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Gradient type (Bio-rad)
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Rehydration of IPG strip

• Standard rehydration solution
• 8M urea, 0.5 CHAPS, 0.2 DTT, 0.5 carrier
  ampholyte, 10 (v/v) glycerol, 0.002 bromophenol
  blue
• Types of rehydration
• Rehydration cassette
• Reswelling tray
• Rehydration loading
• Cup loading

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1. Rehydration cassette

• Disadvantages
• high volume of rehydration solution needs.
• cassette leaking due to urea and detergent.
• rehydration loading of different sample is not
  possible.

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2. Reswelling tray

• Rehydration volume must be controlled.
• 7cm 125 mL
• 13 cm 250 mL
• 18 cm 340 mL
• 24 cm 450 mL

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In reswelling tray

• Rehydration volume is too big.
• Preferable reswell of LMW compounds.
• Leave detergent and HMW compounds outside.
• Over-swelling causing background smearing.
• Rehydration volume is too small.
• Pore size will be too small for HMW proteins to
  enter.
• Rehydration must perform at RT.
• (urea might crystalize at low temperature.)

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3. Rehydration loading

• The sample in lysis buffer diluted with
  rehydration solution.
• Rehydration occurs in an individual strip holder.
• The dry gel matrix takes up the fluid together
  with the protein.
• Small molecules go into the gel matrix faster.
• Proteins diffuse into the fully hydrated gel
  later.
• It takes up to 12 hours for rehydration loading.
• IEF is preformed with the gel surface down.

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IEF sample loading
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4. Cup loading

• The strip is pre-rehydrated with rehydration
  solution. (6 hours)
• The sample is applied into a loading cup at a
  defined pH.
• The proteins are transported into the strip
  electrophoretically.
• IEF is preformed with the gel face up.

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Rehydration loading v.s. cup loading
Rehydration loading
Cup loading
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Comparison between rehydration and cup loading

• Cup loading
• Pro
• Extreme pH condition still works
• Faster entry of protein, less protein-protein
  interaction
• More protein spots developed
• Con
• Protein with pI near application point tends to
  aggregrate
• Does not always work well in all conditions

• Rehydration loading
• Pro
• No precipitation
• Less manipulation
• Higher sample loading
• High entry of HMW protein
• Con
• Protein loss for pH 6-9 or 6-11
• Rehydration time is too long
• Protein might aggregate during rehydration
• Protein with low solubility might precipitate
  inside the gel

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Cover fluid

• Paraffin oil is widely used.
• Cover fluid can prevent
• 1. Drying of the strip
• 2. Crystallization of the urea
• 3. Uptake of O2 and CO2
• Silicon oil is not recommended

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Run IEF step 1
1. Remove protective film from Immobiline
DryStrip gel.
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Run IEF step 2
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2 . Apply rehydration solution to the Strip
Holder.
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Run IEF 3
3. Wet entire length of IPG strip in rehydration
solution by placing IPG strip in strip holder
(gel facing down).
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Run IEF 4
4. Gently lay entire IPG strip in the strip
holder, placing the end of IPG strip over
cathodic electrode.
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Run IEF 5
5. Protein sample can be applied at sample
application well following the rehydration step
if the protein sample was not included in the
rehydration solution.
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Run IEF 6
6. Carefully apply DryStrip Cover Fluid along
entire length of IPG strip.
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Run IEF 7
7. Place cover on strip holder.
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Run IEF 8
8. Place assembled strip holder on Ettan
IPGphor platform
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Temperature

• Spot positions of certain proteins can vary
  dependent on temperature (Gorg. A. 1991)
• Running at 20C is optimal.
• Above the temp where urea might crystalize.
• Below the temp which cause carbamylation.
• Active temperature control is necessary.

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Electric conditions

• Current 50-70 mA/strips
• The strips should never be pre-focused.
• At the beginning of IEF, using low voltage to
  avoid sample aggregation and precipitation or
  overheating.
• Example of voltage program(see below)

Step and hold
Gradient
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V I R

• The set current can limit the achievable voltage,
  when ..
• the sample contains too much salt, or
• buffers are included in the rehydration or lysis
  solution.

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Volthours

• Amount of voltage applied over a certain time
• Example
• 8000 Vh (8kVh) 8000 V in 1 hour or
  4000 V in 2 hours
• The Vh definition corrects the running condition
  of different conductivities in different strips.
• Voltage gradients (ramping) improves the result
  considerably. In order to apply comparable
  voltage loads, the Vh value is a good
  measurement.??

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IEF condition

• Ideal condition no horizontal streaking in 1D.
• The reasons for horizontal streaking
• Overloading effects
• Too short focusing time
• Too long focusing time
• Oxidation of cysteine.
• To much salt, nucleic acid, lipid
• Etc.

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Underfocusing vs Overfocusing

• They all cause horizontal streaks.
• Underfocusing insufficient focusing Vh.
• Overdoucusing too much focusing time. (not
  voltage)
• Negative effects on overfocusing
• Cysteine oxidation.
• Some protein become unstable and being modified.
• (pI change and start moving again..streak)
• Basic narrow gradients are sensitive to
  overfocusing
• Best results are obtained with a focusing phase
  as short as possible at a voltage as high as
  possible.