The document discusses the study of amino acids' properties, focusing on their pKa values, isoelectric points, and how titration curves can be used to analyze them. It details the 2D-PAGE technique for separating and identifying proteins based on their isoelectric point and molecular mass, highlighting its advantages in handling complex biological samples. Additionally, the document addresses challenges in protein analysis, including molecular weight limitations and reproducibility issues, while introducing 2D-DIGE as an advanced method to improve sensitivity and resolve gel variations.

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