shihchung@ntu.edu.tw

02/19 02/20

1

02/26 02/27

2 P1

03/04 03/05

3 X

03/11 03/12

4 P2

03/18 03/19

5 P3/S2

03/25 03/26

6 S2

04/01 04/02

7 P4

04/08 04/09

8 S3

04/15 04/16

9

04/22 04/23

10

04/29 04/30

11

05/06 05/07

12

05/13 05/14

13

05/20 05/21

14 N1 DNA

05/27 05/28

15 N2

06/03 06/04

16 N3

06/10 06/11

17

http://homepage.ntu.edu.tw/~shihchung/

A1

Poster size

90 cm X 120 cm

(1) (2)

b88608043@ntu.edu.tw

520

#3Trypsin

*Trypsin

Trypsin

A B C D

0

5

10

15

20

25

30

1 2 3 4 5

*

50

60

80

90

100

(mg/mL)

0421

BCX P1

OO

O

OO

OO

O

H

H

H H

H

H

HH

H HH+

NN

NN

N

N

N

N

NCCCC

C C

CC C

C

CC C

C

CC C C O

O -R

R

R

R

R

R

R

R

COO-

R group

H N-C-C-N-C-C-N-C-C O O O

CH3N+

Peptide bond1

UV absorbance

Bradford dye-binding protein assay

Biuret protein assayLowry protein assayBicinchoninic acid (BCA) protein asay

Amido black protein assayused in criminal investigations to detect blood present with latent fingerprints

o-phthalaldehyde (OPA) protein assay

2

Specific Binding Group

M

280 nm(aromatic)

UV Absorbance

Fe

Metal

Trp

Heme

RO

O

O

OO

OO

O

H HH+

NN

NN

N

N

N

N

NCCCC

C C

CC C

C

CC C

C

CC C C O

O -

R

R

R

R

R

R

R R

CNH

206 nm(carbonyl)

Phe 260nmTyr 275nmTrp 280nm

Max. absorbance

Peroxidase

3

Bradford Method

Coomassie Brilliant Blue G-250Coomassie Brilliant Blue G-250

CBG 470 nm 595 nm

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.4

-N=N- -NH--OH -SO3-

SO3-

SO3-Bradford dye-binding

280 nm(aromatic)

UV Absorbance

-N=N- -NH--OH -SO3-

SO3-

SO3-

Trp

Arg

RO

O

O

OO

OO

O

H HH+

NN

NN

N

N

N

N

NCCCC

C C

CC C

C

CC C

C

CC C C O

O -

R

R

R

R

R

R

R RCC

CCHN NH2

CNH2+

CNH

206 nm(carbonyl)

5

Biuret methodBiuret peptide bonds biuret biuret 550 nm

Lowry method (Folin-phenol ) Biuret Method peptide bonds nitrogen TyrTrpCys Folin reagent phosphomolybdic-phosphotungstic acid () 660 nm 10-20 Biuret method 10-100

Bicinchoninic acid (BCA) protein asay Biuret BCA 562 nm BCA Folin reagent

6

Lowry et al. (1951). Protein measurement with the Folin-Phenol reagents. J. Biol. Chem. 193: 265-275.

Smith et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85

O C

NH2

NH2 OC

HN

H2N

OC

H2N

UreaBiuret

180 CO

O C

NH

NH2

O C

NH2

O C

NH

NH2

O C

NH2

OC

HN

H2N

OC

H2NCu2+

O C

NH

NH2

O C

NH2

Biuret method

O C

H N

C R

O C

H N

C R

OC

HN

CR

OC

HN

CR

Cu2+

Peptide bondPro

tein

7

Lowry method (Folin-phenol ) Biuret Method peptide bonds nitrogen TyrTrpCys Folin reagent phosphomolybdic-phosphotungstic acid () 660 nm 10-20 Biuret method 10-100

Bicinchoninic acid (BCA) protein asay Biuret BCA 562 nm BCA Folin reagent

8

Lowry et al. (1951). Protein measurement with the Folin-Phenol reagents. J. Biol. Chem. 193: 265-275.

Smith et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85

Biuret method

o-phthalaldehyde (OPA) protein assay

+ +

2-MercaptoethanolPrimary amineOPA

Excitation 340nm Emission 455nm

9

Alcohol dehydrogenase

-amylase

Bovine serum albumin

Carbonic anhydrase

Catalase

-Chymotrypsin

Cytochrome C

-Galactosidase

Hemoglobin (bovine)

Histones

Lysozyme

Myoglobin

Ovalbumin

Ovomucoid

Pepsin

Ribonuclease

Trypsin inhibitor (soy)

Transferrin

Trypsin

Average

5.8

6.8

9.7

8.8

7.6

9.4

25.7

9.5

16.2

9.7

10.4

13.7

10.2

7.8

9.8

11.8

9.1

8.5

11.4

10.2

5.0

6.0

8.4

8.9

6.3

11.6

11.3

9.9

8.3

9.2

12.6

7.9

10.1

8.3

12.4

15.9

10.3

9.0

15.5

9.8

7.8

8.3

21.1

13.0

9.7

7.8

25.3

7.9

19.9

15.8

9.9

20.7

9.4

19.9

4.1

5.3

6.1

12.6

4.9

12.1

Assay Results (protein mg/mL)

Biuret Lowry Bradford

Modified from Bio-Rad

10

Bradford dye-binding

Modified from Bio-Rad

11

Modified from Pierce

12

Protein concentration (g/mL)

BCA Protein Assay

A 5

62 n

m

IgG

BSA

Urea, 6 MGlutathione

Triton X-100, 0.1%Glucose

Tris, 2 MFructose

Sodium phosphateFormic acid, 1 M

SDS, 0.1%Ethanol

Phosphate, 1 MEGTA, 0.05 M

Phenol, 5%EDTA, 0.1 M

NaCl, 5 MEagle's MEM

MgCl2, 1 MDTT, 1 M

Mercaptoethanol, 1 MATP, 1 mM

KCl, 1 MBoric acid

HEPES, 0.1 MAmmonium sulfate, 1 M

Glycine, 0.1 MAcetone

Glycerol, 99%Acetate, 0.6 M

Reagents Compatible With Bio-Rad Bradford Dye-BindingProtein Assay

Derived from Bio-Rad

13

Biuret

Lowry

A 280nm

A 205nm

Bradford

0.05-5.0 mg

0.05-0.5 mg

0.05-2.0 mg

0.01-0.05 mg

0.01-0.05 mg

(Tris )

carbonyl group

BCA 0.02-0.5 mg

14

General Guidelines for Good Pipetting

15

CHOM (chicken egg white ovomucoid)

11% of all egg white proteins

28 kDa, 186 a.a. (predicted MW: 20 kDa)

SDS-PAGE 34~49 kDa

Carbohydrate content: 20~25%

5 N-linked sugar chains

Stable to digestion and heat

Cooked eggs can cause allergic reactionsin CHOM-specific allergic patients

Adapted from IAAI 2005; 136: 23-32

11697

6655

3731

22

kDa

CHOM

16

SDS-PAGE

Trypsin (EC 3.4.21.4)

23.3 kD (porcine)Serine protease

Endopeptidase

Cleaves proteins at the carboxyl side of lysine or arginine, except when either is followed by proline

QGALFKVNRLGWRYGAKPEM

QGALFK VNRLGWR

YGAKPEM

17

TCA

CHOM

TCA TCA

P1

P2

P3