MODERN METHODS in

BIOCHEMISTRY
PROTEIN MODIFICATION
PROTEIN CROSSLINKING
PROTEIN STAINING
ANTIBODY MODIFICATION
IMMUNOPRECIPITATION
METABOLIC LABELLING

PROTEIN
MODIFICATION

PROTEIN MODIFICATION
A. Cys: SH- reagents
B. Lys, Arg
C. Met
D. His
E. Glu, Asp
F. Tyr
G. Trp
H. Ser

A. CYSTEINE : thiol reagents

example of application

A. CYSTEINE
SH- reagents
a) reducing agents

-Mercaptoethanol
DTT = dithiothreitol,
Clelands Ragent)
DTE = dithioerythritol

A. CYSTEINE
SH- reagents
b) oxydising agents
performic acid

 A. CYSTEINE
SH- reagents
c) bloquage de SH
Idoacetate

N-Ethylmaleimide

p-mercuribenzoate

A. CYSTEINE
SH- reagents
d) Ellmanns Reagent
DTNB = Dithionitrobenzoic acid
dosage de ponts -S-S-

B. LYSINE & ARGININE

a) Dosage de NH2- :

FDNB (fluoronitrobenzic acid = ractif de Sanger)

PITC (phenyl-isothiocyanate = ractf dEdman)
DANSYL
DABSYL
anhydride succinique
o-phtalaldehyde

b) Blocage de NH2- :
Fmoc
Citraconic ac

c) Ajouter un site de clivage la trypsine

Cys+Bromoethylamine

FDNB & TNB

PITC (Edmans Reagent)

DANSYL & DABSYL Chloride

Boc & FMoc

ETTF & Succinic anhdydride

ARGININE
Phenylglyoxal & Butanedione

C. METHIONINE
BrCN
clivage spcifique !!!

D. HISTIDINE
Diethylpyrocarbonate
Iodoactate

E. GLUTAMATE & ASPARTATE

diazomethane, carbodiimides (ex. EDAC)

F. TYROSINE
A. Iodination Radioiodination
Labelling with I2+ iodoperoxidase
Radioactive labelling (with 131I2)
Fluorescent labelling
Solid phase liquid phase

B. Adjonction dun groupe Tyr

HO-Phe-Ethylmaleimide

C. Nitrosilation: Tetranitromethane

G. TRYPTOPHANE
BNKS-Skatole
DNP-sulfenylchloride
N-Bromosuccinimide

H. SERINE
DIFP = di-isopropylfluorophosphate
PMSF = phenyl-metyl-sulfonylfluoride

p.ex.:
bloquer les Ser-protases

AA Labelling
Ninhydrin

MODERN METHODS in
BIOCHEMISTRY
PROTEIN MODIFICATION
PROTEIN CROSSLINKING
PROTEIN STAINING
ANTIBODY MODIFICATION
IMMUNOPRECIPITATION
METABOLIC LABELLING

PROTEIN
CROSSLINKING

PROTEIN CROSSLINKING
Protein Cross-linking : Principles and examples
Carbodiimides, Maleimides et Succinimides
(NHS) and related Cross-linking
Bifunctional Double Agents
Homo- or hetero-bifunctional agents, (non-)cleavable

Photoactivatable reagents
Multifunctional and Triple Agents
Trifunctional Cross-linking

Affinity chromatography : matrices preparation

preparation
detection of reactive groups

Biotinylation

A. Protein Cross-linking :
Principles and examples

Buts:
1. Rticuler ensembles 2 proteines
Exemple :biotinylation

2.

Fixer une activit enzymatique sur

une protine.
Anticorps-POD, Anticorps-Phase

3. Attacher une protine sur un support

solide
fabrication dlectrode enzymatique

4. Attacher une protine sur un gel en vue

dune chromatographie daffinit
5. Dterminer linteraction dune protine
avec dautres macromolcules
(protines, polysaccharides) du milieu
6. Etc

A---B

PROTEIN CROSSLINKING
Protein Cross-linking : Principles and examples
Carbodiimides, Maleimides et Succinimides
(NHS) and related Cross-linking
Bifunctional Double Agents
Homo- or hetero-bifunctional agents, (non-)cleavable

Photoactivatable reagents
Multifunctional and Triple Agents
Trifunctional Cross-linking

Affinity chromatography : matrices preparation

preparation
detection of reactive groups

Biotinylation

 Carbodiimides
p.exemple :

1-Ethyl-3-(3-dimethyl)aminopropyl-carbodiimide
(EDAC) - soluble
288
/bs[[1
[1
[2
]d
gr
1
0
[0
end
5740
6185
5020
4300
6049
3910
132
4134300
107
26520
5150
4304
5051
5319
1 I198
6520
5740
6185
5020
3855
7472
2433
455740
6520
4304]
5150]
4528
4793
124 6520]
113
DSt
6130
3993
5
2065050
5319
chemdict
2{bs2527
7568
e g Bd}repeat
begin
4792 ]SP Db
4527

288
/bs[[2
[1
[2
]d
gr
1
0
[0
end
5740
6185
5020
4300
6049
3910
132
4034300
107
26520
5150
4304
5051
5319
1 I198
6520
5740
6185
5020
3855
7472
2433
45
5740
6520
4304]
5150]
4528
4793
124 6520
113
DSt
6130
3993
5
20
4504
65050
5319
chemdict
2{bs
6238
7568
2527
e g 5536
Bd}repeat
begin
47926238
4527
]SP Db
]

%w
userdict/chemdict
L/gr/grestore
L/tr/transform
xl
lpp
SA
RA}{6
-1
st}b/OrA{py
-8
py
np
gs
fill
e
o
cp
a
wy
dp
0
In
x
5
dp/cY
2
0
1
-1
cw
py
bW
gCopyRight
dp
ChemDraw
p
cm
fill
8
DA}{cw
sc
pA
gr
pp}{sqrt
-1
0
m2
2
p
ix
o
dv
1l-9.6
r2
lt{1
-2
m
mv
sc
180
p
s2
cm
px
HA}{dL
0
a}b/PT{8
gr}{pp}{gs
-1
dp
sm
exec}{al
dv
s{dp
dv
m
g
mv
dv
rO
dp
-.6
dv
x-1
py
0
exec
WI
sc
sm
neg
b2
12
neg
st
lp
CA
np
5p
wF
py
n
0mv
at
bd
1.2
LB
sc
p
gr
m
px
neg
p
mv
a}{ex
-9.6
p
2.25
1
st
e
1986,
OA}{1
parcn
p
l2
e
m
lLaser
clip}b/Ct{bs
sl
neg}if/py
l0
p
np
n
pp
L/gs/gsave
gs
gr
OB
mv
aL
at
cp
L/xl/translate
ex
p
lst}b/HA{lW
m
512
0
1
mv
3.375
wy
p
o
px
mv
ro
16.8
SA
gs
1
-1
90
8
py
0
fill
ac
n
np
n/ex
1
llp
st}{0
counttomark{bs
145
-1
OB/bL
1987,
sg
bW
py
ix
aR
dp
wx
px
bd
sc
0.3
Prep
DA}{dL
rro
0.6
px
ac
8
gr
rad
1
sc
fill
dv
cv
p
SA
0
aL
dp
m1
n
m1
0
x
at
DLB
dict
0
eq{DD}{DS}ie
py
end}b/Db{bs{dp
1
xlbd
8
1.2
120
p
-9.6
ey
ne{bW
0
gr
p
neg
px
py
DA}{cw
Cambridge
dp
m
sc
x
mt
rot
-1
rO
l}for
m/w
m
rad
m
L/ie/ifelse
mv
1.5
p
bW
-1
cm
put
n/ey
p
180
py
0
2CA
0
ro
1
0
b1
b1
e
L/S{sf
g
ac
21.6
lp
0
dv
lt{-1
0
-1
SA
180
ac
0
-.6
dp
0
sm
cpt
st
bs
tr/dy
lchemdict
x
OA}{1
2
12
a}ie}b/WW{gs
px
py
a}ie}b/BW{wD
aA
5
p
cp
ne{bW
x0
gr}{gs
np
DA}{dL
8
sc
dv}{bd}ie
m
rot
m
3
-8
e
2.2
st}{Asc
xl
dx
0
6
py
0
p
1
cm
sm}b/dA{[3
DLB
-1
g
5
a
0
sl
360
-1
Scientific
sc
180
16
lx/dx
sc
24.6
gn/dx
-1
L/ix/index
2
lp
px
-1
sqrt
0
p
px
2.25
sm
rgs
12
neg
type[]type
2
rneg}if/px
4
p
mv
0
1
ac
gs
ssc
0
arc
rspy
arc
begin/version
dv/bd
LB
gi
mv
1
SA
o
3
-4.8
x
st}{0
dp
2
OB
rO
180
x
wF
0.5
90
0
SA
0.6
ac
1
CB
e
0
al
p
eq{DB}{DS}ie
gs
dy
gs
DA}{2.25
py
0
rev{neg}if
w
rac
neg
lW
lComputing,
wD
1
m
CA
S]}b/dL{dA
DA}{cw
pp
16
begin
sg
cw
25.8
e
6
CB
rO
px
0.5
2.2
1
n/dy
2
sc
x}if
lp
px
cW
s-1
2
L/l/lineto
sg
x
OA}{1
m
o
0
fill
begin
e
p
0
-2
eq{dp
1
e
np
py
dv
sg
np[{py
p
16
0
cX
np
xl}{xl
mv
cv
rp
dp
fill
0
setgray
agr
bs
x
l0
dv
sbW
lp
5
lSA
fill
wy
0
1
cm
ne
1
-1.6
lx
0arc
0
cp
gr
pcm
sc}b/Ov{OrA
m
-1
0
e
p
bs
o
ac
1
rlineto
23
0OA}{1.5
rO
gr
lly
pp}{2
py
0
pp
wx
o
dp
g/wb
mv
sm
cm
n
sl
16
w
ac
Inc.
p
sc
g
gs
e
sm
px
ne{bW
p
cY
27
gs
L/mt/matrix
p
ac
def/b{bind
1.5
mv
px
clippath
2
g
pp}ifelse
0
cm
at
270
st
ldiv
1
sm
lcm
lp
m
ne
2
WI
st}{Asc
cm
eq{gs
1.6
3
0
1
4
0
py
gr}b/OB{/bS
x
cp
mt
SA
bL
4
sm
slW
0
e
SA
w
gi
st
dup
p
st}{px
sm
bs
AA}{1
or{4
psm
0
gi
27
rO
2
0
fill}b/SA{aF
pDA}{270
ro
lbd
gr
p
al
lOA}{1
m
0
st}]e
p
e
dv/bd
py
l1
al
dp
st
cp
lfill
4.8
ac
2
cp
gs
st
tr
pp
0
ZLB
mv
1
4
g/bb
rad
0.4
1
put
gr
pp
lW
S
rO
-1
def}bind
fill
p
wy
m
1
gr
px
r0
-1
cm
DT}]o
39
lt{pp
25.8
L/mv/moveto
ap
cpt
px
4
S
g
SA
sc
gs
setgray
w
0
o
ac
2
dv
sc
np
end
x}if
py
CA
a/py
-1
x
dv
1sm}b/CB{np[{[{CS
so
setdash}d/cR
dv
x0
p
mv
0
OA}{1
0.5
180
cp
e
wb
round
r}if
g
eq{dL}if
-0.4
3
xl
cv
m
px
2
0
lDA}{180
sc
px
1
0
dp
at
gr}b/In{px
a
exec
rad
ix
st}{1.0
S}if/lp
1
1
4
glW
8
sg
def/L{load
x4
pp
0
ZLB
lW
ro}ie}b/AA{
AA}{1
py
sc
0
3
0
-1
g
nH
Bd
24.6
wx
-0.4
g/cX
-1
rO
fill
w
360
m/aL
ix
p
2
exec}b/C
pp
-2
gr}ie}b/
cm
lp
rgs
1
gr}b/w
sc
ap
o
n
ac
6
a/px
xl
gr
x3
CA
Ac}{0.
ac
o/cX
s{nH
dv
-1
9.6
o
p
sc
4
arc
1
curr
x
2.25
L/m
lp
3
-1
px
lcm
lt{e
lx
gi
wb
12
dx
sg
DA
e
px
-1
sc
LB
p
w
02xa
0
crn

%w
userdict/chemdict
L/gr/grestore
L/tr/transform
xl
lpp
SA
RA}{6
-1
st}b/OrA{py
-8
py
np
gs
fill
e
o
cp
a
wy
dp
0
In
x
5
dp/cY
2
0
1
-1
cw
py
bW
gCopyRight
dp
ChemDraw
p
cm
fill
8
DA}{cw
sc
pA
gr
pp}{sqrt
-1
0
m2
2
p
ix
o
dv
1l-9.6
r2
lt{1
-2
m
mv
sc
180
p
s2
cm
px
HA}{dL
0
a}b/PT{8
gr}{pp}{gs
-1
dp
sm
exec}{al
dv
s{dp
dv
m
g
mv
dv
rO
dp
-.6
dv
x-1
py
0
exec
WI
sc
sm
neg
b2
12
neg
st
lp
CA
np
5p
wF
py
n
0mv
at
bd
1.2
LB
sc
p
gr
m
px
neg
p
mv
a}{ex
-9.6
p
2.25
1
st
e
1986,
OA}{1
parcn
p
l2
e
m
lLaser
clip}b/Ct{bs
sl
neg}if/py
l0
p
np
n
pp
L/gs/gsave
gs
gr
OB
mv
aL
at
cp
L/xl/translate
ex
p
lst}b/HA{lW
m
512
0
1
mv
3.375
wy
p
o
px
mv
ro
16.8
SA
gs
1
-1
90
8
py
0
fill
ac
n
np
n/ex
1
llp
st}{0
counttomark{bs
145
-1
OB/bL
1987,
sg
bW
py
ix
aR
dp
wx
px
bd
sc
0.3
Prep
DA}{dL
rro
0.6
px
ac
8
gr
rad
1
sc
fill
dv
cv
p
SA
0
aL
dp
m1
n
m1
0
x
at
DLB
dict
0
eq{DD}{DS}ie
py
end}b/Db{bs{dp
1
xlbd
8
1.2
120
p
-9.6
ey
ne{bW
0
gr
p
neg
px
py
DA}{cw
Cambridge
dp
m
sc
x
mt
rot
-1
rO
l}for
m/w
m
rad
m
L/ie/ifelse
mv
1.5
p
bW
-1
cm
put
n/ey
p
180
py
0
2CA
0
ro
1
0
b1
b1
e
L/S{sf
g
ac
21.6
lp
0
dv
lt{-1
0
-1
SA
180
ac
0
-.6
dp
0
sm
cpt
st
bs
tr/dy
lchemdict
x
OA}{1
2
12
a}ie}b/WW{gs
px
py
a}ie}b/BW{wD
aA
5
p
cp
ne{bW
x0
gr}{gs
np
DA}{dL
8
sc
dv}{bd}ie
m
rot
m
3
-8
e
2.2
st}{Asc
xl
dx
0
6
py
0
p
1
cm
sm}b/dA{[3
DLB
-1
g
5
a
0
sl
360
-1
Scientific
sc
180
16
lx/dx
sc
24.6
gn/dx
-1
L/ix/index
2
lp
px
-1
sqrt
0
p
px
2.25
sm
rgs
12
neg
type[]type
2
rneg}if/px
4
p
mv
0
1
ac
gs
ssc
0
arc
rspy
arc
begin/version
dv/bd
LB
gi
mv
1
SA
o
3
-4.8
x
st}{0
dp
2
OB
rO
180
x
wF
0.5
90
0
SA
0.6
ac
1
CB
e
0
al
p
eq{DB}{DS}ie
gs
dy
gs
DA}{2.25
py
0
rev{neg}if
w
rac
neg
lW
lComputing,
wD
1
m
CA
S]}b/dL{dA
DA}{cw
pp
16
begin
sg
cw
25.8
e
6
CB
rO
px
0.5
2.2
1
n/dy
2
sc
x}if
lp
px
cW
s-1
2
L/l/lineto
sg
x
OA}{1
m
o
0
fill
begin
e
p
0
-2
eq{dp
1
e
np
py
dv
sg
np[{py
p
16
0
cX
np
xl}{xl
mv
cv
rp
dp
fill
0
setgray
agr
bs
x
l0
dv
sbW
lp
5
lSA
fill
wy
0
1
cm
ne
1
-1.6
lx
0arc
0
cp
gr
pcm
sc}b/Ov{OrA
m
-1
0
e
p
bs
o
ac
1
rlineto
23
0OA}{1.5
rO
gr
lly
pp}{2
py
0
pp
wx
o
dp
g/wb
mv
sm
cm
n
sl
16
w
ac
Inc.
p
sc
g
gs
e
sm
px
ne{bW
p
cY
27
gs
L/mt/matrix
p
ac
def/b{bind
1.5
mv
px
clippath
2
g
pp}ifelse
0
cm
at
270
st
ldiv
1
sm
lcm
lp
m
ne
2
WI
st}{Asc
cm
eq{gs
1.6
3
0
1
4
0
py
gr}b/OB{/bS
x
cp
mt
SA
bL
4
sm
slW
0
e
SA
w
gi
st
dup
p
st}{px
sm
bs
AA}{1
or{4
psm
0
gi
27
rO
2
0
fill}b/SA{aF
pDA}{270
ro
lbd
gr
p
al
lOA}{1
m
0
st}]e
p
e
dv/bd
py
l1
al
dp
st
cp
lfill
4.8
ac
2
cp
gs
st
tr
pp
0
ZLB
mv
1
4
g/bb
rad
0.4
1
put
gr
pp
lW
S
rO
-1
def}bind
fill
p
wy
m
1
gr
px
r0
-1
cm
DT}]o
39
lt{pp
25.8
L/mv/moveto
ap
cpt
px
4
S
g
SA
sc
gs
setgray
w
0
o
ac
2
dv
sc
np
end
x}if
py
CA
a/py
-1
x
dv
1sm}b/CB{np[{[{CS}{CS}{cB}{CW}{pp}{cB}{CW}{pp}{cB}]o
so
setdash}d/cR
dv
x0
p
mv
0
OA}{1
0.5
180
cp
e
wb
round
r}if
g
eq{dL}if
-0.4
3
xl
cv
m
px
2
0
lDA}{180
sc
px
1
0
dp
at
gr}b/In{px
a
exec
rad
ix
st}{1.0
S}if/lp
1
1
4
glW
8
sg
def/L{load
x4
pp
0
ZLB
lW
ro}ie}b/AA{np
AA}{1
py
sc
0
3
0
-1
g
nH
Bd
24.6
wx
-0.4
g/cX
-1
rO
fill
w
360
m/aL
ix
p
2
exec}b/CS{p
pp
-2
gr}ie}b/Cr{0
cm
lp
rgs
1
gr}b/wD
sc
ap
o
n
ac
6
a/px
xl
gr
x3
CA
Ac}{0.5
ac
o/cX
s{nH
dv
-1
9.6
o
p
sc
4
arc
1
currentmatrix
x
2.25
L/m/mul
lp
3
-1
px
lcm
lt{e}if
lx
gi
wb
12
dx
sg
DA}{120
e
px
-1
sc
LB
p
w
021.6
xaL
0
cm
rnal
def}b/d/def
mv
dv
xp
fill
sm
120
ne{py
2
py
SA
d/wF
gs
s/wx
r0
gs
5
edp/cY
Ac}{1.0
1
18
mv
pp
m
spp
sm
0
n
dp
g/cY
rad
gr
cX
rO
OA}{1
2
9.6
1
st}{0
aR
3mv
360
rO
0
AA}{1
w
dict
dv
DS
L/n/neg
sg
1
cvi/nSq
0
CA
bd
cm
gs
x
1.5
0
cY
dp
dy
sn
0
16.8
ac
r0
p
s/wy
x
p
fill
px
m
gr}{dp
0
np
sg
p
e
DA}{90
rad
m
d/WI{wx
xl
Ov}{0.5
sm
-1
p
rlineto
lsbb
pp
ap
o
d/aF
0
p
-1
lscw
np
px
lgr
lp
wF
fill
arc
np
w
iX
0
cv
o
mv
L/a/add
sc
0st
180
4
mv
lW
xsc
x0
a
o
0
cm
lW
2
dx
py
eq
gr
L/np/newpath
1.5
9.6
m
g
px
gr
bs
10
0
bL
st}b/DS{np
py
o
at
p
px
m
CA
90
sl
px
cX
1st}b/Asc{OrA
scW
g
Ov}{Asc
o
p
180
o
lp
sm
mv
gs
py
eSA
ro
nSq
d/aR
dx
dp
12
0
4.0
rO
dv/m1
py
3
o
lAA}]e
DA}{3
iY
lW
eq
gac
p
lp
0.3
2
p
eq{2
dp
st}{Asc
OA}{1
ne{wy
rad
L/al/aload
8
n
ac
2
6
eq
pap
setmiterlimit
2
m
mv
bb
py
px
8
p
4
-1
l0.25
m
1
cp
m
dv
cm
4.8
and{pp
RA}{3
g
a
lgi
LB
0p
py
5
xsc
4
cp
p
pp
In
n
e
rexec
dv
sl
cm
-1
pa
lal
py
ggi
a
mv
LB
dy
p
gs
dp
sm
4.8
0
px
2
p
d/aA
sp
lst}b/Ar1{gs
pp}b/DT{gs
rO
cY
arc
dp
L/pp/pop
1
sc
l}for
pp
gs
al
0
sm
m1
dv
lHA}{1
1
gs
sm
p
lp
st}{gs
gr}b/ac{arcto
sl}b/cB{12
27
lx
cv
12
a
1
pp
eq
wx
sg
sc
pp
cm
L/at/atan
4lgs
px
a
1
st}{rO
SA
spy
ex
px
45
st}b/DD{gs
cm
cp}b/ZLB{LB}b/Ar{dp
dv
-1
0
nSq{.135
np}b
fill
cp}{p
and{bb
sqrt
sg
0DT}{pp}ie}ie}{pp}ie}forall}b/I{counttoma
1
dx
cm
sm
-1
py
OA}{270
360
lW
sm
bW
p
d/nH
dp
g
rsm
fill
gr
ly
smv
sc
3
lp
sm
1
s/b1
st
dp
py
L/r/roll
2
end
5
np
sc}b/LB{9
dv/m1
l}ie}repeat
cm
ey
0
-1
gr
arc
sst
270
gr}b/CA{aF
ac
dv
1
OB
p
bS
px
ne{bW}{5
2
6
g
st
L/cp/closepath
PT
gr}{pp}{DB}{gs
gs
srsm
mv
e
x}if
g
px
d/o{1
a
gs
.667
dL
4{pp}repeat}b/pA
gr}b/RA{lW
CA
3
p
s/lx
0
dv
CA
cm
2
bb
cm
rO
ix
l0
p
py
0.5
st}{Asc
xDS
rO
OA}{180
4
lx
L/ro/rotate
ex
dp
DA}{1
p
l3
wy
a
sm
x.865
gi
sm
-6
px
0
ix}b/rot{3
rO
gr}b/DB{gs
mv
ix
sg
n/ex
g}{bb
p
pp
s/ly
lW
lW
ne{ly
al
m1
lW
st
mv
lxl
py
st
lp
xl
fill
rO
0
st}b/SP{gs/sf
LB
2
-1
pp
39
gr}b/Bd{[{pp}{[{DS}
m}ie
.667
gr
3
x
s0
m/aL
p
m
x
CA
m/w
wx
21
4
gr
lW
-1
gs
py
sc
0
12
py
5
lx
lt{Ar1}{gs
ley
np
lt{pp
g
L/cv/curveto
cm
bW
px
OA}{120
-1
cm
-10
180
dv/m2
m
0.5
sl
1
-2
rL/sc/scale
lp
bb
px
-1
OB
n/ey
-0.4
xs12
0
lp
0
32
rs/b1
sm
ac
sm
2np
3
dv
py
aL
5
r}b/x{e
lW
sg
27
rcurveto
exec}{al
CA
np
0sdv
OB
1d/rO{4
g}ie
cm
p
cw
-0.4
py
x
rad
a
1
rO
st
fill
2
-8
r0
DA}{1
x5
x/lW
x}if
mv
wF
CA
dx
py
aR
bW
em}if/
gr
ly
lW
1
sm
p
ac
bW
gr
cY
27
sw
rOA
n/
lm
dp
lW
d}
ey
ZL
lx
px
sL/
o0
2
p
o
cp
lp
sL
.x
0
m
-

Maleimide et Succinimide (NHS)

O

currentpoint 192837465

O
currentpoint 192837465

PROTEIN CROSSLINKING
Protein Cross-linking : Principles and examples
Carbodiimides, Maleimides et Succinimides (NHS) and
related Cross-linking

Bifunctional Double Agents

Homo- or hetero-bifunctional agents,
(non-)cleavable
Photoactivatable reagents
Multifunctional and Triple Agents
Trifunctional Cross-linking
Affinity chromatography : matrices preparation
preparation
detection of reactive groups
Biotinylation

Strategies
Homobifunctional
Non-Cleavable
Cleavable

Heterobifunctional
Non-Cleavable
Cleavable

Photoactivatable (azidophenacyl,
Varia: tri-functional,etc

Bifunctional Double Agents

Ia. non cleavable Homobifunctional
HOMOBIFUNCTIONAL REAGENTS
1,2-bis-Maleimidoethane
Short, sulfhydryl reactive homobifunctional crosslinking reagent. Crosslinks
to 9.9 .
Maleimide group labels sulfhydryls at pH 6.5-7.5.

Bifunctional Double Agents

Ia. non cleavable Homobifunctional
BMH : 1,6-bis-Maleimidohexane
Sulfhydryl reactive homobifunctional crosslinking reagent.
Ref. . Partis, M.D., et.al. (1983) J. Prot. Chem. 2, 263-277.

Bifunctional Double Agents

Ia. non cleavable Homobifunctional
1,4-Phenylene dimaleimide
Antibody-enzyme conjugation.
Ref. Knight, P., et.al. (1979) Biochem. J. 179-191.; 2. Yoshitake, S., et.al.
(1979) Scand. J. Immunol. 10, 81

Bifunctional Double Agents

Ib. cleavable Homobifunctional
MMP : Maleimidopropionic acid maleimidomethyl ester
Useful for crosslinking to erythrocyte membranes.
Cleavable with 0.02 M hydroxylamine or mild base.

Ref. Sato, S., et.al. (1981) J. Biochem. 1177-1185

Bifunctional Double Agents

Ib. cleavable Homobifunctional
DTME : Dithio-bis-maleimidoethane
Sulfhydryl reactive and cleavable homobifunctional crosslinking reagent.
Cleavable with DTT, DTE, or TCEP.
Ref. Han, J.C., et.al. (1994) Anal. Biochem. 220, 5-10.

Bifunctional Double Agents

IIa. Non cleavable Heterobifunctional
Maleimidoacetic acid-NHS
Ultra short, sulfhydryl and amino reactive heterobifunctional
crosslinking reagent.
Ref. May, J.M. (1989) Biochemistry 28, 1718.; 2. Sayre, L.M., et.al. (1984) J. Med. Chem. 27, 1325.

Bifunctional Double Agents

IIa. Non cleavable Heterobifunctional
2-Maleimidoethyl amine
Water soluble! Derivatize C-terminal peptides with EDCHCl.
Introduces maleimide group into glycoproteins (via Nakane coupling).
Ref. Nakane, P.K. (1975) Ahn. N.Y. Acad. Sci. 254, 203

Bifunctional Double Agents

IIa. Non cleavable Heterobifunctional
3-Maleimidopropionic acid NHS
Short, sulfhydryl and amino reactive heterobifunctional
crosslinking reagent.
Wide application in immunodiagnostics.

Ref. Kitagawa, T. (1981) Enz. Immunoassay, 81.

Bifunctional Double Agents

IIa. Non cleavable Heterobifunctional
6-Maleimidocaproic acid (2-nitro-4-sulfo) phenyl ester
Water soluble! Quantitate reaction with amines by release of 2-nitro 4-sulfo
phenol.
Ref. Aldwin, L., Nitecki, D. (1987) Anal. Biochem. 164, 494.

Bifunctional Double Agents

IIa. Non cleavable Heterobifunctional
3-Maleimidopropionic acid
Sulfhydryl reactive heterobifunctional crosslinking reagent. For
preparing peptide-protein conjugates.
Ref. 1. Rich, D., et.al. (1975) J. Med. Chem. 18, 1004.; 2. Moroder, L., et.al. (1983) BioPolymers 22, 481.

Bifunctional Double Agents

IIa. Non cleavable Heterobifunctional
SIA : Succinimidyl iodoacetate
Sulfhydryl and amino reactive heterobifunctional protein
crosslinking reagent.
Ref. Eur. J. Biochem. (1984) 140, 63.; 2. Rector, E.S., Sehon, A., et.al. (1978) J. Immun. Meth. 24, 321.

Bifunctional Double Agents

IIa. Non cleavable Heterobifunctional
SBA : Succinimidyl bromoacetate
Sulfhydryl and amino reactive heterobifunctional crosslinking reagent.
Sulfhydryl selectivity at neutral or slightly alkaline pH is comparable to
iodoacetamide, and perhaps greater than maleimide.
Ref. Cuatrecasas, P., et.al. (1969) J. Biol. Chem. 244, 4316.; 2. Bernatowicz, M.S., et.al. (1986) Anal. Biochem.
155, 95.

Bifunctional Double Agents

IIb. Cleavable Heterobifunctional
SATA : N-succinimidyl S-acetylthioacetate
Reacts with primary amines to add protected sulfhydryls.
Thioacetyl group can be deprotected with 0.02 M hydroxylamine
hydrochloride to render free sulfhydryl.
Ref. Duncan, R.J.S., et.al. (1983) Anal. Biochem. 132. 68-73

Bifunctional Double Agents

IIb. Cleavable Heterobifunctional
SATP : N-succinimidyl S-acetylthiopropionate
Reacts with primary amines to add protected sulfhydryls. Extended analog of
SATA.
Thioacetyl group can be deprotected with 0.02 M hydroxylamine hydrochloride
to render free sulfhydryl.
Ref. Duncan, R.J.S., et.al. (1983) Anal. Biochem. 132. 68-73.

Bifunctional Double Agents

IIb. Cleavable Heterobifunctional
PDA : Pyridine dithioethylamine hydrochloride
Sulfhydryl amination reagent. Water soluble!
Wide use in diagnostics, and conjugation chemistry.
Quantitate thiol groups by the release of 2-thiopyridone
max=343 nm. =8,080 M-1 cm-1.
Ref. Johnson, R.J., et.al. (1985) J. Biol. Chem. 260, 7161-7164

Bifunctional Double Agents

IIb. Cleavable Heterobifunctional
SPDP hydrazide
Sulfhydryl and carbohydrate reactive derivative of SPDP. DTT or
TCEP cleavable.
Useful for preparing carbohydrate linked immunoconjugates.
Examles : conjugating antibodies to nerve growth factor (NGF).
Ref. 1. Zara, J.J., et.al. (1991) Anal. Biochem. 194, 156-162.; 2. Friden, P.M., (1993) Science 159, 373-378

Bifunctional Double Agents

IIb. Cleavable Heterobifunctional
LC-SPDP
Sulfhydryl and amino reactive heterobifunctional protein
crosslinking reagent.
Extended analog of SPDP with similiar properties and uses.
Ref. Carlsson, J., et.al. (1978) Biochem, J. 173, 723-737

Bifunctional Double Agents

IIb. Cleavable Heterobifunctional

SPDP
Sulfhydryl and amino reactive heterobifunctional protein crosslinking reagent.

Protein thiolation reagent.

Tethering to artificial membranes.
Preparing antibody-toxin conjugates.
Hapten preparation.
Sulfhydryl modification can be followed by release of 2-thiopyridone,

max=343 nm. =8,080 M-1 cm-1.

Ref. 1. Carlsson, J., et.al. (1978) Biochem. J. 173, 723-737.; 2. Walden, P., et.al. (1986) J. Mol. Cell Immunol. 2, 191-197.; 3. Cumber, A.J., et.al. (1985)

Meth. Enzymol. 112, 207-225.; 4. Gordon, R.D., (1978) PNAS 84, 308-312

Photoactivatable reagents
4-(N-Maleimido)benzophenone
Sulfhydryl reactive photocrosslinking reagent.
Generates photoactivatable conjugates from thiols.
Ref. 1. Agarwal, R., et.al. (1991) J. Biol. Chem. 266, 2272.; 2. Leszyk, J., et.al. (1988) Biochemistry 27, 6983.; 3. Tao, T., et.al. (1984) Biophys. J. 45, 261.

Photoactivatable reagents
p-Azidobenzoic acid (99+%)
Useful for generating photoaffinity probes for mapping receptor-ligand
complexes.
Introduces carboxylate functionality onto non-derivatizable surfaces, ie.
polystyrene.
Ref. Chatreneet, B., et.al. (1990) Proc. Natl. Acad. Sci. USA 87, 3378.

Photoactivatable reagents
HSAB : N-Hydroxysuccinimidyl 4-azidobenzoate
Amino and photoreactive protein crosslinking reagent.
example : photoaffinity crosslinking of calmodulin to binding proteins.
Ref. 1. Vandlan, R.L., et.al. (1985) J. Biol. Chem. 260, 10889-10892.; 2. Wood, C.L., et.al. (1985) J. Biol. Chem. 260, 1243-1247.; 3.
Goewert, R.R., et.al. (1982) Biochemistry 21, 5310.

Photoactivatable reagents
ABH : p-Azidobenzoylhydrazide
Photoreactive heterobifunctional crosslinking reagent.
Useful for crosslinking glycoproteins and antibodies.
Ref. 1. O'Shannessy, D.J., et.al. (1985) J. Applied Biochem. 7, 347-355.; 2. O'Shannessy, D.J., et.al. (1984) Immunol. Lett. 8, 273-277.

Photoactivatable reagents
3-Nitro-4-fluorophenylazide
Heterobifunctional crosslinking reagent for photoaffinity labeling.
Ref. 1. Hagedorn, M., et.al. (1978) J. Org. Chem. 43, 2070-207; 2. Tometsko, A.M., et.al. (1976) Int. J. Peptide Protein Res. 8, 331.

Photoactivatable reagents
p-Azidophenacyl bromide
Sulfhydryl and photoreactive crosslinking reagent.
Ref.: Schwartz, I., Ofengand, J. (1974) Proc. Natl. Acad. Sci. 71, 230.

Photoactivatable reagents
p-Azidobenzoyl-[2-(2-pyridyldithio)ethyl amide]
Converts sulfhydryls into cleavable photoaffinity probes.
Used to label granulocyte C5a receptor.
Ref. Johnson, R.J., Chenoweth, D.E. (1985) J. Biol. Chem. 7161-7164.

Photoactivatable reagents
SAND : sulfo-Succinimidyl 2-[m-azido-onitrobenzamido]ethyl-1,3-dithiopropionate
LC, water soluble, and cleavable analog of ANB-NOS.

Multifunctional and Triple Agents

Trifunctional Cross-linking
Mal-3 : tris-(2-Maleimidoethyl)amine
Sulfhydryl reactive tris-maleimide reagent for preparing trimeric
aggregrates of polypeptides.

Multifunctional and Triple Agents

Trifunctional Cross-linking
TSAT, NHS-3 : tris-Succinimidyl aminotriacetate
Amino reactive trifunctional crosslinking reagent.
Amino core for preparation of dendritic molecules or aggregrates.
Can be selectively aminated to generate mixed trimers.

Multifunctional and Triple Agents

Trifunctional Cross-linking
SDMB : Succinimidyl-3,5-dimaleimidophenyl benzoate
Amino and sulfhydryl reactive trifunctional protein crosslinking reagent. Useful
for generating multivalent antitumor antibody fragments.
Ref. Schott, M.E., et. al. (1993) Bioconjugate Chem. 4, 153-165.

Multifunctional and Triple Agents

Trifunctional Cross-linking
Mal-4 : tetrakis-(3-Maleimidopropyl)pentaerythritol
Tetrafunctional and sulfhydryl reactive dendritic core for preparing clusters.
Useful for generating multivalent ligand complexes.

RADIO-IODINATION
b-(4-Hydroxyphenyl)ethylmaleimide
Sulfhydryl reactive. Introduces 4-hydroxyphenyl ligand.
Iodinate with Iodogen or iodobeads.

RADIO-IODINATION
sulfo-Succinimidyl-3(4-hydroxyphenyl)propionate
Water soluble Bolton-Hunter reagent!

Biotinylation

(+)-Biotin
Vitamin H
Essential cellular growth factor.

K =10-15 M!!!

Specific and high afinity binding to AVIDIN :

D
Wide application in immunology, diagnostics, affinity chromatography, etc.
Used to label DNA.
Ref. 1. Wagner, A.F.; Folkers, K. (1964) Vitamins and Coenzymes 138-159.; 2. Green, N.M. (1975) Adv. Protein Chemistry 29,85-133.; 3. Roffman,
E., et. al. (1986) Biochem. Biophys. Res. Comm. 136, 80.; 4. (1992) Acta Pharm. Fenn. 101, 147.

Biotinylation
sulfo-Succinimidyl (+)-Biotin
Water soluble and amino reactive biotinylation reagent.
Wide use in immunodiagnostics.
Ref. LaRochelle, W.J., et.al. (1986) J. Immunol. Methods 92, 65-71

Biotinylation
(+)-Biotin-PDA
Sulfhydryl reactive biotinylation reagent..
Quantitate biotinylation by release of 2-thiopyridine,
lmax343 nm, e=8,080 M-1 cm-1.
Ref. 1. Ghebrehiwet, B., et.al. (1988) J. Immunol. Methods 110, 251-260.; 2. Carlsson, J., et.al. (1978) Biochem. J. 173, 723-737.

Biotinylation
(+)-Biotin-X-X-sulfo-NHS LC-LC-sulfo-NHS-(+)-Biotin
Water soluble! Extra long chain length.

ANTIBODY MODIFICATION
Biotinylation - HABA-avidin
HRP = horse radish peroxidase
AP = alcaline Phosphatase
GOD = glucose oxydase
-Galactosidase

ANTIBODY MODIFICATION
Fluorescence labeling
Fc removal
Amines
SH-groups
Carbohydrate groups

Radioactive labeling
iodination of Tyr

PROTEIN MODIFICATION
w. IMPACT TWIN

IMPACT-TWIN System

protein labeling, ligation and cyclization

The IMPACT-TWIN Protein Fusion and Purification System utilizes the inducible
selfcleavage activity of engineered protein splicing elements (termed

INTEINS) for protein purification and manipulation.

Produces a target protein with an N-terminal cysteine and/or a C-terminal
thioester for protein labeling, ligation and cyclization.

Contains three expression vectors that allow the fusion of a target protein to modified
mini-intein tags.
Isolation of proteins with a C-terminal thioester for protein ligation or with an Nterminal residue other than Met
The protein ligation reaction allows the incorporation of non-coded amino acids or
labels into a protein sequence.
Furthermore, pTWIN vectors can be used to fuse a target protein to the Cterminus of intein1, which can be induced to undergo cleavage by a
temperature/pH shift. This is advantageous for purifying target proteins that are
sensitive to reducing agents.
Insertion of the target protein between two intein tags allows the generation of
circular protein species with a peptide bond at the site of ligation

IMPACT-TWIN System
protein labeling, ligation and
cyclization

Advantages
* Single-column purification--no additional steps to remove the affinity tag
* Flexibility of fusion to either the C-terminus or N-terminus of the target protein
maximizes the probability of successful expression and purification
* Yields native amino acid sequence
* Release of fusion partner without the use of proteases
* Isolation of proteins with or without an N-terminal methionine residue
* Use of either thiol reagent or pH and temperature shift to induce on-column cleavage
* The only system that produces proteins possessing an N-terminal cysteine and/or Cterminal thioester for use in protein labeling, ligation and cyclization
* IPTG-inducible T7 Promoter (6) for highlevel expression and tight regulation of
expression
The System Includes:
* Three pTWIN E. coli expression vectors: pTWIN1, pTWIN2, pTWIN-MBP1
* Chitin Beads: Affinity matrix used for fusion protein purification (binding capacity=2 mg/ml).
* E. coli Strain ER2566
* Anti-CBD Serum (rabbit)
* Ssp DnaB Intein Forward, Mxe Intein Reverse and Mth RIR1 Intein Reverse Sequencing Primers
* DTT and SDS-PAGE Sample Buffer
* Instruction Manual

Protein splicing: inteins

16-10

Inteins were discovered less than 10 years ago

Inteins represent the protein equivalent to the genomic DNA intron:
elements that are spliced out of the final (mature) product
unlike introns, inteins are self-splicing through the use of an endonuclease
used commercially in biochemical applications (explanation on board)
- inteins are 134-608 aa
- the mini-inteins do not have the
endonuclease domain but have
other characteristics of inteins

Legend:
Schematic illustration of protein splicing (upper part) and intein
structure (lower part). The two terminal regions of the intein
sequence form the splicing domain of a typical bifunctional
intein. Six conserved sequence motifs (A to G) are shown. The
intein sequence begins with the first amino acid of motif A and
ends with the second last amino acid of motif G. Motifs C and E
are the dodecapeptide motifs of endonuclease. A star (*) stands
for hydrophobic amino acids (V, L, I, M). A dot (.) stands for a
nonconserved position.
adapted from Liu, X.-Q. (2000) Annu. Rev. Gen. 34, 61.

Protein splicing: inteins

Legend:
Scenarios of intein evolution. A, loss of the
endonuclease domain. B, breaking the intein
sequence. C, loss of the splicing function. D,
replacing the C-extein with a cholesterol
molecule (green dot). E, loss of C-terminal
cleavage and splicing. F, loss of N-terminal
cleavage and splicing. G, placing intein
fragments on two ends of a protein. H,
breaking intein into 3 fragments. I, separating
a middle fragment of the intein from the rest.
J, presence of two different split inteins.
Scenarios A to D are based on examples
observed in nature.
Scenarios E to G are based on engineered
artificial inteins.
Scenarios H to J are purely hypothetical.

16-11

Intein protein splicing: mechanism

N-extein represents the
N-terminal polypeptide
segment that is retained
C-extein represents the
C-terminal segment that is
retained
the Intein is what is
spliced out (much as a
genomic DNA intron)
Cys1, Asn154 and
Ser155 represent
conserved residues
involved in the splicing
reaction

16-12

Figure 1: Protein
cyclization.
The target protein is
fused between Intein1
and Intein2 and released
with an N-terminal
cysteine and a Cterminal thioester.

Figure 2: Protein
purification by pH
and temperature
induced cleavage:
Using a pTWIN
vector, the intein
tag is fused to the
N-terminus of the
target protein.
The target protein
is then released by
pH and
temperature
induced cleavage.

Figure 3: Protein
purification by thiolinduced cleavage:
Using a pTWIN
vector, the intein tag is
fused to the Cterminus of the target
protein.
The target protein is
then released by thiolinduced cleavage.