2D-PAGE & DIGE

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 pKavalues
•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 pIvalue.
•The Ionizablegroups 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. •Thismethodisusedfortheseparationandidentificationof
proteinsinacomplexmixtureusingtwoseparatedimensionsthat
arerunperpendiculartooneanother.
•Basedontwoproperties(IEFandmolecularmass)
•Thisallowsacomplexbiologicalsampletobeseparatedovera
largerarea,increasingtheresolutionofeachcomponent.
Why 2D-PAGE?

9. •IPGstripsareused.
•ImmobilizedpHgradient(IPG)gels.
•IPGsaretheacrylamidegelmatrixco-polymerizedwith
thepHgradient,whichresultincompletelystable
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. •Detectseparatedproteinsbystainingor
immunodetectionafterblottingontoamembrane.
•Powerfultoolsandtechniquesareusedtocomparethe
samples&identifytheproteinofinterest.
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.Molecularweights:
•Protein>250kDadonotenter2
nd
SDS-PAGEproperly.
3.Reproducibility:
•Variationmostcomesfromsamplepreparation.
4.Lesssensitivity(lowerresolution)
Challenges for 2-DE

20. •Twodimensionaldifferenceingelelectrophoresis
•2D-DIGEisanadvancedversionofclassicaltwo-dimensionalgel
electrophoresis(2D-PAGE).
•Theproteinsamplesarelabeledwithfluorescentdyesandthen
separatedby2D-PAGE.
2D-DIGE

21. Steps Involved

23. •SolvestheGel-to-gelvariationsproblemin2DPAGE,
byenablingthemultiplesamplesinsinglegels
•Sensitivityimprovedduetouseoffluorescentdyeand
laser.
Advantages

24. THANK YOU