Download - 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 NAIDUMsc 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
MALDI-TOF MSTandem mass spectrometer (MS/MS)cDNA microarray
Celera Genomics Inc.
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Proteomics solution
IEF
SD
S-P
AG
E
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2-Dimension Electrophoresis (2-DE) for Protein Separation
Speaker: C. C. WuDate: 31/10/2001
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.
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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.
Net
cha
rge
-3
-2
-1
0
1
2
3
2 3 4 5 6 7 8 9 10 11
pH
Isoelectric point
NH3+
COOH
NH3+
COOH
pH < pIPositive charge
NH3+
COO-
NH3+
COO-
pH = pI
NH2
COO-
NH2
COO-
pH > pINegative charge
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sample
pH 9 -
pH 3 +
Isoelectric focusing(1st dimension)
General principle and protocol of 2-Dimension Electrophoresis
MW
pH gradient
SDS-PAGE
Ampholytes
polyacrylamide
2nd dimension
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Traditional Equipment for Isoelectric focusing (IEF):
Ampholytespolyacrylamide
Cathode (-) electrode solution
Anode (+) electrode solution
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Traditional 2-Dimensional Electrophoresis
Anode (+) electrode solution
Cathode (-) electrode solution
Disadvantage: cathodic driftA
mph
olyt
epo
lyac
ryla
mid
e
pH 3 pH 3 pH 3
pH 9 pH 7 pH 5
Time
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Immobilized pH Gradient (IPG)
Polyacrylamide gel
Acidic buffering group:
Basic buffering group:CH2 = CH-C-NH-R
O
COO-
NH3+
Acrylamide monomer
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Gradient maker
plastic support film
Production of Immobilized pH Gradient (IPG) strip
A
C
B
F
E
Dacid
ic
basi
c
pH 3
pH 10
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IPGphor (IEF System)Amersham Pharmacia Biotech Inc.
Protein IEF CellBio-Rad Laboratories
Equipment for Isoelectric focusing (IEF):
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Lysis solution:8M Urea4% NP-40 or CHAPS40mM Tris base
Sample preparation
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
2-DE sample Rehydration solution
Rehydration solution:8M Urea2% NP-40 or CHAPS2% IPG buffer (Ampholyte)0.28% DTTTrace Bromophenol blue
IPG strip holder
Position the
IPG strip
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IPG strip rehydration and sample loading
Strip holder
Cathode (-) electrode
Anode (+) 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
Time
Vol
tage
Holder cover
IPG strip
Electrode
Electrode pads
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Second dimension: SDS-PAGE• SDS equilibration• SDS-PAGE
SDS equilibration buffer50 mM Tris-HCl6 M Urea30% Glycerol2% SDSTrace Bromophenol
SD
S
SDS-PAGE SDS-PAGE
0.5% agarose in running buffer
SDS-PAGE
Marker in paper
IPG strip
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Protocol of silver stain:
50% methanol25% acetic acid4hr
ddH2O x 3 times
30min/time
0.004% DTT solution30min
0.1% AgNO3
30min
ddH2O
30 sec
3% Na2CO3
0.0185% formaldehyde
2.3M citric acid
5% acetic acid25% methanol
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2-DE separation of soluble E. coli proteins
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