Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)

1. Two Dimensional
Polyacrylamidegel
Electrophoresis (2D-PAGE)
Supervised by-
Dr. SuhasSutarBharat
Presented by-IPSITA SAHOO
Reg. no-200705180160
DOMAIN-
Genomics And Genetic
Engineering

2. CONTENTS
Introduction; 2D PAGE
Workflow of 2D PAGE
1D; Isoelectricfocusing
2D; SDS-PAGE
Visualization of results; staining
Advantages vs. Disadvantages
Reference

3. 2D-PAGEstandsforTwoDimensionalPolyacrylamidegelElectrophoresis
2D-PAGEisaformofgelelectrophoresisinwhichseparationandidentificationof
proteinsinasamplearedonebydisplacementin2dimensionsorientedatrightangles
tooneanother(orthogonal).
2D-PAGEisawidelyusedmethodfortheanalysisofcomplexproteinmixture
extractedfromcells,tissues,orotherbiologicalsamples.
ThistechniqueswasfirstdevelopedbyO’FarrellandKlosein1975.
2D-PAGEisusedin3sequentialsteps-

4. Work flow of 2D

7. 1.
2.

8. Using2D-PAGE,100–1000’sof
polypeptidescanbeanalyzedin
asinglerun.
Theproteinscanbeseparatedin
pureformfromthespots.The
spotscanbequantifiedandalso
analyzedbyMS.
Inthismethodpolypeptidescan
beprobedwithantibodiesand
alsotheycanbetestedforpost-
translationalmodifications.
2D-PAGEhaslimiteddynamic
range.
Thistechniqueincludealarge
amountofsamplehandling,less
reproducibility.
Itisalsonotautomatedforhigh
throughputanalysis.
Difficultytoseparatelow
abundanceproteins,acidicand
basicproteins,verylargeand
verysmallproteinsand
hydrophobicproteins.
Advantages and Disadvantages

9. References
Beranova-Giorgianni,S.(2003)Proteomeanalysisbytwo-dimensionalgel
electrophoresisandmassspectrometry:strengthsandlimitations.TrACTrendsin
AnalyticalChemistry22(5),273-281.
StephenJFey*andPeterMoseLarsen(2001)2Dornot2D.CurrentOpinionin
ChemicalBiology2001,5:26–33.
WayneFPatton*,BirteSchulenbergandThomasHSteinberg(2002)Two-dimensional
gelelectrophoresis;betterthanapokeintheICAT?.CurrentOpinioninBiotechnology,
13:321–328.
DavidE.Garfin(2003)Two-dimensionalgelelectrophoresis:anoverview.Trendsin
AnalyticalChemistry,Vol.22,No.5
http://www.weihenstephan.de/blm/deg/manual/manualwork2html02test.htm
http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html