2D-PAGE is a 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. 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.

1. 2D-PAGE & DIGE
Nixon Mendez
Department of Bioinformatics

2. • Study the acidic and basic properties of amino acids
• Determine pKa values
• To recognize the unknown amino acid.
Titration curve of amino acids

3. • 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.

4. Titration Curve for
glycine:
The shaded boxes,
centered at about
pK1=2.34 & pK2 = 9.60
indicates the regions of
greatest buffering
power.

6. • 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

7. • 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.

8. • 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?

9. • 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

10. 1. SAMPLE PREPARATION
2. RUN FIRST DIMENSION – IEF
3. RUN SECOND DIMENSION – SDS-PAGE
4. VISUALIZE AND ANALYZE
How to perform 2D-PAGE?

12. Immobilized strips are dehydrated which allows rehydration of the
strips directly with the sample to be separated.

15. • Detect separated proteins by staining or
immunodetection after blotting onto a membrane.
• Powerful tools and techniques are used to compare the
samples & identify the protein of interest.
4. Visualize And Analyze

16. Image analysis

17. Image analysis

18. 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

19. 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

20. • 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

21. Steps Involved

23. • 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

24. THANK YOU