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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 . Speaker: Dr. J. S. Yu Date: 3/21/2002. The age of X-omics and biotechnology:

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slide1

Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry for protein identification

  • 2-Dimensional Gel Electrophoresis
  • MALDI-TOF Mass Spectrometry

Speaker: Dr. J. S. Yu

Date: 3/21/2002

slide2

The age of X-omics and biotechnology:

  • Genomics: Human genome project
  • Transcriptomics: cDNA microarray
  • Proteomics:
  • Development and involvement of mass spectrometry

Celera Genomics Inc.

cDNA microarray

Tandem mass spectrometer (MS/MS)

MALDI-TOF MS

slide4

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

slide5

NH3+

NH3+

NH2

COOH

COO-

COO-

NH3+

NH3+

NH2

COOH

COO-

COO-

pH < pI

Positive charge

pH = pI

pH > pI

Negative charge

Isoelectric point

Net charge

pH

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.

slide6

General principle and protocol of 2-Dimension Electrophoresis

Ampholytes

sample

Isoelectric focusing

(1st dimension)

pH 9 -

pH 3 +

polyacrylamide

2nd dimension

SDS-PAGE

MW

pH gradient

slide7

Traditional Equipment for Isoelectric focusing (IEF):

Ampholytes

polyacrylamide

Cathode (-) electrode

solution

Anode (+) electrode solution

slide8

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

slide9

CH2 = CH-C-NH-R

O

Immobilized pH Gradient (IPG)

Acrylamide monomer

Acidic buffering group:

COO-

Basic buffering group:

NH3+

Polyacrylamide gel

slide10

acidic

basic

Production of Immobilized pH Gradient (IPG) strip

Gradient maker

A

D

plastic support

film

B

E

C

F

pH 3

pH 10

slide11

Equipment for Isoelectric focusing (IEF):

IPGphor (IEF System)

Amersham Pharmacia Biotech Inc.

Protein IEF Cell

Bio-Rad Laboratories

slide12

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

slide13

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

slide14

IPG strip rehydration and sample loading

Strip holder

Anode (+)

electrode

Cathode (-)

electrode

30 voltage 12hr

slide15

Electrode

pads

Holder cover

IPG strip

Electrode

Voltage

Time

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

slide16

SDS

SDS-PAGE

SDS-PAGE

Marker in paper

0.5% agarose

in running buffer

SDS-PAGE

  • 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

slide17

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