-
B2
Enzyme Analysis
1
a.
b.
(albumin)
(immunoglobulin)
c.
1.1 Biuret
carbonyl carbonyl biuret ( mg) Tris Biuret BCA (bicinchoninic
acid)
1.2 Lowry
biuret Folin-Ciocalteau phosphomolybdic-phosphotungstate ( 0.1
mg)
1.3 UV
ECX 2005 139
a. 280 nm 200 nm 280 nm
-
B2
(molar extinction coefficient)
b. E (1%, 280 nm) 4~15 ( 10) E 10 280 nm 1 1 mg/mL E b c
(c % 100 mL b 1 cm)
c. E 10 280 nm 1 1 mg/mL
1.4 Coomassie Blue (dye binding) Bradford Method
Coomassie Brilliant Blue G-250 (CBG) 595 nm ( g)
1.5
peroxidase heme 403 nm ()
1.1 (-N-C-C-N-C-C-) (Lys, Arg, Tyr )
140 ECX 2005
NC C N
N
N
CC
C CN
CC C
CNCC N C
CNCC
N CC
O
O
O
OO
O
OO
O
R
R R
R
R
R
H
H
H
H
H
H
H
H
HH
H+
Cu2+
OH...phsophomolybdic-phosphotungstate
Lowry Method
Biuret Method
UV Absorbance
280 nm
206 nm(carbonyl)
(aromatic)
CC
CCCNH3
+
LysCC
C
NH2+
Tyr
Coomassie Blue G
HNC
Special Binding Groups (heme)
(carbonyl)
(metal)
Arg
NH2
O
ActiveSite
Fe N=NOH
SO3-
NH
SO3-
SO3-
CuCu
N=N
OH
SO3-
NH
SO3-
SO3-
-
B2
2
2.1
a.
2.1
(1) () --
(2)
(3) ( pH ) ()
(4) ()
(5) ()
b.
(Vmax)Km pH
2.2
(t) (P) P/t (vo)
2.2.1
(t) (P) (P/t)
a.
NAD+ NADH
2.1
NAD+
NADH
10 x Km
ECX 2005 141
-
B2
240 280 320 360 400
340
NA
Ethanol NAD+ Acetaldehyde NADH H+
NADH 340 nm ( 2.2)
b.
(P) (Q) 340 nm
S P Q 2.2 NADH
c.
(invertase, IT)
+ IT
d.
HPLC (PPC)
e. (manometry)
Warburg
f.
pH pH
g. HPLC
HPLC
2.2.2
a. 3~5% TCA () pH
b. (100) 10 min ( RNase)
142 ECX 2005
D
NADH
+
Wave length (nm)
-
B2
c. 1% SDS protease K SDS
d. EDTA
e.
f. (cold)
2.2.3 (continuous-reaction)
a.
340 nm
b.
2.2.4
a.
(Pi)
(Glc)n-Glc + Pi (Glc)n + Glc-1-P () (Glc)n + Glc-1-P (Glc)n-Glc
+ Pi ()
(Pi) Pi
b.
(primer) Glc-1-P
(amylose)
( 2.3 A)
ECX 2005 143
2.3
Glc-1-P
BA
SP
A
SP BAPh
Glucose+Pi
B
-
B2
c.
-amylase (BA) Glc-1-P phosphatase (Ph) ( 2.3 B)
()
2.3
2.3.1
a. pH 2.1
2.1
pH
Formate 3.0 ~ 4.5 Citrate 3.0 ~ 6.2 Acetate 3.7 ~ 5.5 Phosphate
5.8 ~ 8.0 HEPES 6.5 ~ 8.5 Tris 7.1 ~ 8.9 pH Borate 8.1 ~ 9.0
Carbonate 9.7 ~ 10.7 Universal 2 ~ 12 pH
b. ( 2.2)
2.2
144 ECX 2005
NaN3 (sodium azide) 0.01% EDTA, EGTA 0.1~1 mM -Mercaptoethanol
1~10 mM Dithiothreitol (DTT or DTE) 1~5 mM BSA (bovine serum
albumin) 0.1~10 mg/mL Tween-20, Triton X-100 0.5~0.05%
-
B2
Glycerol, glucose 50% Urea 6~8 M PMSF, TPCK, TLCK,
benzamidine
c.
pH pH (Tris)
d.
pH
2.4
2.4 e. Stock solution
pH
2.3.2
a.
b. (aliquot) () -
4
c. (-20) 50%
d.
2.3.3
a.
()
ECX 2005 145
1 mM 10 mM 100 mM 1 M
-
B2
b.
(1) pH
( SDS )
(2) cofactor
cysteine -SH EDTA -SH
(3)
PMSF (phenylmethylsulfonyl fluoride) Ser PMSF
(4)
c.
(1) 4
(2)
(3) BSA
(4) ()
(5)
(6) ()
2.3.4
146 ECX 2005
(activity unit) pH 1 mole
-
B2
3
3.1
3.1.1
a.
(mobility)
( mV) ( )
b.
pH pH pI pI () pH ( 3.1)
c.
pH 8.3 pH pI 8.3
d. ()
pH
3.1.2
( 3.2)
a. (moving-boundary electrophoresis)
ECX 2005 147
++ANODE CATHODE
Friction
Voltage
Charge
- -
76
89
10
543
11
+ -0Net Charge of Protein
Environmental pH
Isoelectric point, pI
3.1 pH
-
B2
- + b. (zone electrophoresis) Cellulose
Protein Denatured
(band)
(1)
() ()
(2) () cellulose acetate
(thin-layer electrophoresis, TLE)
(3)
(starch gel electrophoresis) (polyacrylamide gel
electrophoresis, PAGE) (agarose gel electrophoresis)
c. (1) (isoelectric focusing)
(2) (peptide mapping)
(3) (Western blotting) (immunostainning)
(4) (preparative electrophoresis)
(5) (immunoelectrophoresis)
(6) (capilliary electrophoresis) HPLC
(7) Pulse field gel electrophoresis DNA
3.1.3
a. 100~500V
b. (1620) (810)
c.
3.1
()
(TLE) Cellulose acetate
Partial Denatured
Starch Gel
Serious tailing
148 ECX 2005
3.2
-
B2
3.2
PAGE PAGE
3.2.1 PAGE
a. (disc-PAGE)
Disc-PAGE PAGE
SDS-PAGE
b. SDS (SDS-PAGE)
SDS SDS-PAGE SDS-PAGE (denatured) (native)
c.
pI 8.3 pH SDS -SDS-PAGE
3.2.2 PAGE
3.2.2.1
a. (monomer) (acrylamide)H2C=CH-CO-NH2
Acrylamide Bis
b. (bridge) Bis [N,N'-methylene-bis(acrylamide)]
c. (free radical) (ammonium persulfate, APS) riboflavin (
BB2)
d. TEMED (tetramethylethylenediamine)
3.2.2.2
a.
ECX 2005 149
b.
-
B2
c.
3.2.3 PAGE
3.2.3.1
3.2
pH
1 () Tris-glycine 8.3 -
2 Tris-glycine 8.3 -
3 Tris-HCl 6.9 5%
4
Tris-HCl 8.3 5~20%
5 () Tris-glycine 8.3 -
a. 3, 4 pH (3) pH (pH 6.9, glycine pI) (disc)
b. 3.3 (4) (3) (2) (1) (5) ()
c.
3.2.3.2
( 3.4)
Glycine ( pH > 6.9) ( pH = 6.9 zwitterion)
a. 3.5A
Gly
b. -- pH pH 8.3-6.9-8.9 Gly pI 6.9
150 ECX 2005
Sample
Running gel
+
-
Stacking gel
(1)(2)
(3)
(4)
(5)
3.3
3.4
Glycine: Negative charged No net charge
Chloride ion:
Proteins:
-
B2
c. 3.5B Gly () Gly
d.
e. 3.5CGly
3.2.3.3
3.6 disc-PAGE SDS-PAGE
a. X, Y, Z X > Y > Z pH (8.3) X Y (pI < 8.3) Z (pI >
8.3)
3.3 Mobility
Protein QuaternaryStructure
MolecularMass (D)
pI Native PAGE SDS-PAGE
X Tetramer (40,000)4 5.8 Y Monomer 88,000 5.2 Z Monomer 60,000
9.3
b. native-PAGE X, Y Z Z X (160 kD) Y
c. SDS-PAGE X, Y, Z SDS SDS ()
ECX 2005 151
A CB
+ +
8.3
6.9
8.9
3.5
-
B2
d. SDS X, Y, Z
Z Y
e. X Y Z SDS-PAGE SDS (40 kD) Y Z
f. SDS-PAGE SDS X (160 kD)
3.2.4
a.
ammonium persulfate, TEMED, acrylamide APS APS
b.
Bis acrylamide
c.
() -mercaptoethanol acrylamide
d.
() APS
152 ECX 2005
3.6 Disc-PAGE SDS-PAGE
X Y Z x Y Z+ SDS
+ +
Native-PAGE SDS-PAGE
+-
-
-
B2
e.
pH ()
f.
g.
pH 6.9
h.
()
3.3
3.3.1
3.7 ()
a.
(1) (ammoniacal silver)
CBR
(2) Coomassie Brilliant Blue R-250 (CBR) CBR
Coomassie Blue R-250 G-250
b.
(glycoprotein) (3) PAS (periodic acid-Schiff's) CBR Schiff
c.
ECX 2005 153
UV 300 nm (4)
-
B2
d. KCl
SDS-PAGE 0.3 M KCl 4 15 min (5) SDS KDS
e.
(6)PAGE (disk)
f. (autoradiography)
154 ECX 2005
NC C N
N
N
CC
C CN
CC C
CNCC N C
CNCC
N CC
O
O
O
OO
O
OO
O
C
R R
R
R
H
H
H
H
H
H
H
H
HH
H+ -
CC
CCCNH3
+
Lys CCC
NH2+
Tyr
HNC
Arg
NH2
O
ActiveSite
N=N
OH
SO3-
NH
SO3-
SO3-
Coomassie Blue R
CCC
NH2+
Ammoniacal
silver
Ag
Glutaraldehyde
(Cys)
Ag+H N...2NHGlu
3H N...Ag...NH3+
Lys
OHUV
Absorbance(300nm)
CO H
OH OH
CO H
Periodate
Schiff's reagent
Ammoniacal silver
CN
H
CarbohydrateStaining
Ag
C
N
H
Dye3H N...Ag...NH3
+
Ser O C
Specific Binding Groups (metal,SDS-KCl)
N=N
OH
SO3-
NH
SO3-
SO3-
OO
3.7
-
B2
g.
p.72~74 (gel dryer) (scanner)
3.3.2 (IEF)
Ampholyte pI ampholyte ampholyte pH pI pH (pH = pI) IEF pI
3.3.3
IEFSDS-PAGE
disc-PAGE
3.3.4
a. (nitrocellulose) ( 3.8A ) (nylon)
b. ponceau amido black (immunostaining) ( 3.8B)
c.
ECX 2005 155
B
CoomassieBlue
Staining PonceauStaining
A
+
Gel
-
HRP
Filter Paper
Nitrocellulose
3.8
-
B2
4
4.1
a.
b. (molecular weight marker)
(kD)
Thyroglobulin (669, 330); ferritin (440); catalase (232);
immunoglobulin G (160); lactate dehydrogenase (140); serum albumin
(67); ovalbumin (43); lactalbumin (14.4)
c.
(peroxidase 405 nm heme )
d. Blue Dextran 2000 ( 2,000 kD)void volume (Vo) Blue Dextran
0.1~0.2 M (NaCl)
e.
(1) Vo
(2) HPLC FPLC
4.2
a.
156 ECX 2005
disc-PAGE SDS-PAGE SDS-PAGE
-
B2
b.
pI disc-PAGE SDS-PAGE
c. (marker)
d.
disc-PAGE pI (4~6 )
4.3
4.3.1
a. (S)
b.
4.3.2
a. (cDNA)
b. (post-translational modification) ()
4.3.3
ECX 2005 157
-
B2
5
( Tris )
5.1 N- C-
N-
a. N- C-N- dansylation dansyl HCl dansylation N- ( 5.1)
b. polyamide (TLC plate) dansyl UV 20 dansyl HPLC
c. N- (blocked)
d. ( chymotrypsin)N-
e. C- carboxylpeptidase C-
5.2
a. 6 N HCl 4 N methanesulfonic acid 110 24 hHPLC
b. metallothionein Cys Cys
c. HCl tryptophan glutamine asparagine glutamic acid ( Glx)
aspartic acid ( Asx) ( Tris) HPLC
158 ECX 2005
5.1 dansylation N-
Dansylation
HCl hydrolysis 2-DChromatography
N-terminal
TLC plate
UV
N-
-
B2
5.3
5.3.1 cDNA
(reverse biochemistry) cDNA
GCG ()
a.
(SWISSPRO)
b.
c.
signature signature PEST (KDEL)
d.
5.3.2 Edman
a. Edman
dansylation N- PITC (phenylisothiocyanate) N- PTH Edman N- HPLC
Edman ( 5.2)
ECX 2005 159
5.2 Edman degradation
N-1 2
1
1
2
2
PITC
PTH-
Second cycle
Amino acidanalysis
PTH-
PTH-
N-
Peptide
-
B2
b.
c. (1)
(2)
(3) prosthetic group
(4)
d.
(1)
(2)
()
5.4
a.
( 5.3)
b.
160 ECX 2005
-
B2
5.4.1
a. Trypsin (Lys, Arg); Chymotrypsin (Phe, Tyr, Trp); Sa protease
(Asp, Glu)
b. CNBr (Met)
5.4.2
a. /
90
b. HPLC
HPLC
c. SDS-PAGE
SDS-PAGE
5.5
5.5.1
(280 nm) 1.3
5.5.2
a. X
b. NMR ()
ECX 2005 161
5.3
Proteases Cutting sites
-
B2
6
ELISA
( Harlow E, Lane D (1988) Antibodies,
A laboratory manual. Cold Spring Harbor Laboratory)
6.1
a.
b.
() ( carrier BSA KHL hemocyanin)
c.
() hapten () carrier
d.
() carrier (PC/GENE)
e.
f.
162 ECX 2005
-
B2
6.2
a.
6.1A
(TiterMax)
b.
6.3
a.
ELISA ELISA 5,000 ( 5,000 ELISA 50% )
b.
6.1A pristane NS-1
c.
ECX 2005 163
6.1
BALB/c
2
(Trial Bleeding)
0
4
6
8
NS-1 Cell10
Ascites Fluids12
14wk
spin down cells ()+ 2X mL PBSammonium sulfate (AS) fractionation
0-40% sat.spin down pellet
Pelletresuspended in 40% AS
spin down pellet
Pelletdissolved in X mL PBSdialysis in PBS,three changes
spin down precipitate
IgG (stored in freezer)
+ glycerol (equal volume)Supernatant
X mL (X = 1 - 10)
BALB/c
A
B
(10 cells)6Pristane (0.5 mL)
TiterMax
Freund'sIncomplete Adjuvant
Antigen (50 mg/mouse) 0.5 mL
Freund's Complete Adjuvant
-
B2
d.
(Ig) ( 6.1B)
6.4
a.
b.
(CNBr-Sepharose) ( 6.2)
c.
d.
e. (ELISA)
164 ECX 2005
CNBr-Sepharose
SDS-PAGE
Ab
H
L
Coupling
6.2
-
B2
7
7.1
Central Dogma Central Dogma
(1) 7.1
(2) 7.1 2000
ECX 2005 165
Protein Technology
327
323
Basic Protein Techniques
373
363
Protein Purification
M1220
380
M1230
Protein Analysis
368
375
365
364Proteome
374
Protein Structure & Function
Protein Engineering
Protein Function
Protein Structure
Protein Engineering
Biochemical Engineering
356
7.1
-
B2
(3)
7.2
7.2.1
(what you see, what you get)HPLC
7.2
(1) SDS-PAGE
(2)
166 ECX 2005
7.2
MALDI-TOF
(1) (2)
(3)
(4)
-
B2
(3)
N-
HPLC
(4)
HPLC FPLC LC
7.2.2
(1)
Edman degradation 10~100 pmole
PC/GENE
(2)
LC LC/Mass
7.3
Genomic Project
proteomeProteome
7.3.1 proteome
(1) proteome proteome hexokinase glycolysis TCA cycle
ECX 2005 167
(2)
-
B2
(3)
7.3.2 proteome
(1) (marker protein)
(2)
168 ECX 2005
Blackstock WP, Weir MP (1999) Proteomics: quantitative and
physical mapping of cellular proteins. Trends Biotech 17:
121~127
1 1.1 Biuret 1.2 Lowry 1.3 UV 1.4 Coomassie Blue (dye binding)
Bradford Method 1.5
2 2.1 2.2 2.2.1 2.2.2 2.2.3 (continuous-reaction) 2.2.4
2.3 2.3.1 2.3.2 2.3.3 2.3.4
3 3.1 3.1.1 3.1.2 3.1.3
3.2 3.2.1 PAGE 3.2.2 PAGE 3.2.2.1 3.2.2.2
3.2.3 PAGE 3.2.3.1 3.2.3.2 3.2.3.3
3.2.4
3.3 3.3.1 3.3.2 (IEF) 3.3.3 3.3.4
4 4.1 4.2 4.3 4.3.1 4.3.2 4.3.3
5 5.1 N-C- 5.2 5.3 5.3.1 cDNA 5.3.2 Edman
5.4 5.4.1 5.4.2
5.5 5.5.1 5.5.2
6 6.1 6.2 6.3 6.4
7 7.1 7.2 7.2.1 7.2.2
7.3 7.3.1 proteome 7.3.2 proteome