Electrophoresis

(Part II)

Electrolysis:
Passing of electric
current through an
electrolyte
Choosing a length of IEF Strip
• As the length increases
• Loading capacity increases
• Resolution of proteins increases
• Number of spots detected increases
• Focusing time increases
• Cost effectiveness decreases

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Isoelectric Focusing
• Types:
• Vertical – Slab gel (like SDS-PAGE) OR capillary

• Horizontal – can be liquid ampholytes OR Strips (e.g. 2D-PAGE)

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Isoelectric Focusing - Horizontal Gel
Steps in Horizontal IEF (Manual method)
• Place a few drops of light paraffin oil to adhere the plastic
template to the cooling plate and smear it over the surface.

• Place the template on the cooling plate.

• Make sure there are no air bubbles trapped between the

cooling plate and the template.

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Isoelectric Focusing - Horizontal Gel
Steps in Horizontal IEF (Manual method)
• Place a few drops of paraffin oil on the plastic template. Then
place the gel on top.
• Align the gel carefully on the template again ensuring that no air
bubbles are trapped between the polyester backing of the gel.
• Press the wrapping of the gel to squeeze out air bubbles.
• When there are no more bubbles, remove the wrapping entirely.

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Isoelectric Focusing - Horizontal Gel
Steps in Horizontal IEF (Manual method)
• Carefully place the sample applicators (filter paper 8mmx5mm)
using a pair of forceps on the gel
• Once the applicator has been placed on the gel, DO NOT move it.
• Load 15 microlitres of sample on the applicator with a
micropipette. Load the same amount of standards on the first,
middle and last applicators.
• Soak the electrode strips in the appropriate buffers (Cathodic strip
1M NaOH; Anodic strip 1M H3PO4)

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Isoelectric Focusing - Horizontal Gel
Steps in Horizontal IEF (Manual method)

• Lay the strips at the designated positions on the gel. Excess lengths
should be cut off so that the strip fits within the gel.

• Unscrew the electrode holders and slide them until they are over the
electrode strips then place the electrode plate over the gel. Place the
safety lid back

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Isoelectric Focusing - Horizontal Gel
Dry Strips
• Used for 2D-gel electrophoresis
• Dehydrated gel between two plastic strips so gel needs to be
rehydrated
• Contains immobilized ampholytes for defined pH ranges
• Different lengths available
• Sample is applied with gel face-down or by cup-loading

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Choice of a pH Range + Length

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Isoelectric Focusing - Horizontal Gel
Dry Strips
• Dry gel strips need to be rehydrated with Rehydration
buffer.
• Passive rehydration: Gel strips are put face-down over buffer +
sample 12-15 hrs
• Active rehydration: Gel strips are put face-down over buffer +
sample and a low voltage of 20-120V is applied for 12-15 hrs
• Cover fluid (Paraffin/mineral oil): Prevents evaporation and drying.

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Isoelectric Focusing - Horizontal Gel
Dry Strips
• Dry gel strips need to be rehydrated with Rehydration buffer.
• If the rehydration is done without the sample, then it is usually passive
and the sample added by Cup-Loading.

• Washing with water.

• Place gel facing up in strip holder

• Place sample cup and test for leakage

with rehydration buffer

• Remove rehydration buffer

• Add sample

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Running Conditions for IEF
Per strip: 50 μA , 10,000V generally.

•Typically starts with low voltage and Gradually increased

through a series of steps to the desired focusing voltage

•A longer focusing time is required for:

• Longer length of strip

• Narrow pH range

• Higher protein load

• Higher urea/detergent concentration

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Post-IEF Processing
If just doing IEF
• Horizontal gels
• Fix with trichloroacetic acid and stain with Coomassie OR silver
staining (like SDS-PAGE)
• IPG (Immobilized pH gradient) strips
• Staining with Crystal violet OR Zinc imidazole

If doing 2D-PAGE
• Strips can be stored at -80C for several months if not used immediately.
• When ready to second dimension, MUST be equilibrated.
• Before an IPG DryStrip can be used in SDS-PAGE, it has to be
immersed in buffer containing SDS to allow the focused proteins to
interact with SDS.
• Long equilibration times (10 - 15 min) plus the use of urea and
glycerol improves protein transfer to SDS-PAGE.
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Two-Dimensional Gel Electrophoresis

• 2D gel electrophoresis is
widely used to separate
complex mixtures of
proteins into many more
components than is
possible in conventional
one dimensional
electrophoresis
• O’Farrell 2D Gel System
• Each dimension separates
proteins according to
different properties
• Isoelectric point
• Size
• Each spot in gel is assigned
X,Y coordinates
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O’Farrell 2D Gel System

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Differential Gel Electrophoresis (DIGE)

• DIGE has the same principle as SDS-PAGE.

• It can be done in one-or two-dimensions.

• Additionally DIGE requires fluorescent protein stains (up

to three of these), a gel box, and a gel scanner.

• Fluorescent dyes include Cy2, Cy3 and Cy5 (Amersham

system).

• These have similar sizes and charges, which means that

individual proteins move to the same places on 2-D gels no
matter what dye they are labeled with.

• Detection down to 125 pg of a single protein. 19

Differential Gel Electrophoresis (DIGE)

• Proteins samples are produced by homogenizing cells.

• Standard chemistry links dyes to proteins via lysine side-

chains through Amide bond.

• Linear response to protein concentration over a 105

concentration range.

NHS ester reaction scheme for chemical conjugation to a primary amine. R represents a labeling
reagent or one end of a crosslinker having the NHS ester reactive group; P represents a protein or other molecule
that contains the target functional group (i.e., primary amine).

NHS: N-Hydroxysuccinimide 20