2D-PAGE & DIGE

Nixon MendezDepartment of Bioinformatics

• Study the acidic and basic properties of amino acids

• Determine pKa values

• To recognize the unknown amino acid.

Titration curve of amino acids

• Amino acids in aqueous solution exist predominantly in isoelectric form.

• The characteristic pH at which the net electric charge is zero is called the isoelectric point (pI).

• So, an amino acid has a net negative charge at any pH above its pI, and has a net positive charge at any pH below its pI.

• Each amino acid has its own pI value.

• The Ionizable groups of amino acids act as weak acids or bases, giving off or taking on protons when the pH is altered.

• Titration curves are produced by monitoring the pH of given volume of a sample solution after successive addition of acid or alkali.

Titration Curve for

glycine:

The shaded boxes,

centered at about

pK1=2.34 & pK2 = 9.60

indicates the regions of

greatest buffering

power.

• Proteins are separated according to molecular mass.

• This is good for simple samples.

Drawback:

• Based on only one property

ie. Molecular mass.

1D-Gel electrophoresis

• 2D-PAGE is a form of gel electrophoresis used for

1. Separation

2. Identification

• Principle:

2D-PAGE separates proteins according to their isoelectric point

first and molecular mass.

What is 2D-PAGE ??

of proteins in a complex

biological sample.

• This method is used for the separation and identification of

proteins in a complex mixture using two separate dimensions that

are run perpendicular to one another.

• Based on two properties (IEF and molecular mass)

• This allows a complex biological sample to be separated over a

larger area, increasing the resolution of each component.

Why 2D-PAGE?

• IPG strips are used.

• Immobilized pH gradient (IPG) gels.

• IPGs are the acrylamide gel matrix co-polymerized with

the pH gradient, which result in completely stable

gradients

2D-PAGE

1. SAMPLE PREPARATION

2. RUN FIRST DIMENSION – IEF

3. RUN SECOND DIMENSION – SDS-PAGE

4. VISUALIZE AND ANALYZE

How to perform 2D-PAGE?

Immobilized strips are dehydrated which allows rehydration of the

strips directly with the sample to be separated.

• Detect separated proteins by staining orimmunodetection after blotting onto a membrane.

• Powerful tools and techniques are used to compare thesamples & identify the protein of interest.

4. Visualize And Analyze

Image analysis

Image analysis

1. Spot number:

• 10,000-150,000 gene products in a cell.

• PTM makes it difficult to predict real number.

• It’s impossible to display all proteins in one single gel.

Challenges for 2-DE

2. Molecular weights:

• Protein > 250 kDa do not enter 2nd SDS-PAGE properly.

3. Reproducibility:

• Variation most comes from sample preparation.

4. Less sensitivity (lower resolution)

Challenges for 2-DE

• Two dimensional difference in gel electrophoresis

• 2D-DIGE is an advanced version of classical two-dimensional gel

electrophoresis (2D-PAGE).

• The protein samples are labeled with fluorescent dyes and then

separated by 2D-PAGE.

2D-DIGE

Steps Involved

• Solves the Gel-to-gel variations problem in 2D PAGE,

by enabling the multiple samples in single gels

• Sensitivity improved due to use of fluorescent dye and

laser.

Advantages

THANK YOU