MALDI-TOF

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• Matrix-Assisted Laser Desorption Ionization
  Time-of-Flight (MALDI-TOF) Mass spectrometry for
  protein identification
• 2-Dimensional Gel Electrophoresis
• MALDI-TOF Mass Spectrometry

M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D
Research scholar.
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• The age of X-omics and biotechnology
• Genomics Human genome project
• Transcriptomics cDNA microarray
• Proteomics
• Development and involvement of mass
  spectrometry

Celera Genomics Inc.
MALDI-TOF MS
Tandem mass spectrometer (MS/MS)
cDNA microarray
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Proteomics solution
IEF
SDS-PAGE
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2-Dimension Electrophoresis (2-DE) for Protein
Separation
The core technology of proteomics is 2-DE At
present, there is no other technique which is
capable of resolving thousands of proteins in one
separation procedure.
Speaker C. C. Wu Date 31/10/2001
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Isoelectric point (pI) Isoelectric point is the
pH of a solution at which the net charge of
protein is zero. In electrophoresis there is no
motion of the particles in an electric field at
the isoelectric point.
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General principle and protocol of 2-Dimension
Electrophoresis
sample
Isoelectric focusing (1st dimension)
Ampholytes
pH 9 - pH 3
polyacrylamide
2nd dimension
SDS-PAGE
MW
pH gradient
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Traditional Equipment for Isoelectric focusing
(IEF)
Ampholytes polyacrylamide
Cathode (-) electrode solution
Anode () electrode solution
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Traditional 2-Dimensional Electrophoresis
Disadvantage cathodic drift
Cathode (-) electrode solution
pH 9 pH 7
pH 5
Ampholyte polyacrylamide
pH 3 pH 3
pH 3
Time
Anode () electrode solution
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Immobilized pH Gradient (IPG)
Acidic buffering group
COO-
Acrylamide monomer
Basic buffering group
NH3
Polyacrylamide gel
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Production of Immobilized pH Gradient (IPG) strip
Gradient maker
A
D
plastic support film
B
E
C
F
pH 3 pH 10
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Equipment for Isoelectric focusing (IEF)
IPGphor (IEF System) Amersham Pharmacia Biotech
Inc.
Protein IEF Cell Bio-Rad Laboratories
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Sample preparation
Lysis solution 8M Urea 4
NP-40 or CHAPS 40mM Tris base
Cell line
Lysis solution
Sonication
vacuum
Lysis solution
Centrifugation
Measurement of protein
2-DE sample
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IPG strip rehydration and sample loading
Rehydration solution
2-DE sample
Rehydration solution 8M Urea 2
NP-40 or CHAPS 2 IPG buffer
(Ampholyte) 0.28 DTT Trace
Bromophenol blue
IPG strip holder
Position the IPG strip
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IPG strip rehydration and sample loading
Strip holder
Anode () electrode
Cathode (-) electrode
30 voltage 12hr
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First dimension Isoelectric focusing
1. Place electrode pads (?) 2. 200 V
step-n-hold 1.5hr 3. 500 V
step-n-hold 1.5hr 4. 1000 V gradient
1500vhr 5. 8000 V gradient (?)
36000vhr
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• Second dimension SDS-PAGE
• SDS equilibration
• SDS-PAGE

SDS equilibration buffer 50 mM Tris-HCl 6
M Urea 30 Glycerol 2
SDS Trace Bromophenol
IPG strip
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Protocol of silver stain
50 methanol 25 acetic acid 4hr
ddH2O 30 sec
ddH2O x 3 times 30min/time
3 Na2CO3 0.0185 formaldehyde
0.004 DTT solution 30min
2.3M citric acid
0.1 AgNO3 30min
5 acetic acid 25 methanol
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2-DE separation of soluble E. coli proteins
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THANK YOU