BTN Protocol Guide-08

Bio-Rad Laboratories
| Membranes and Blotting
Life Science Group
Western Analysis
Molecular Weight Estimation a
2000
nd Q
Alfr
u
ed Nobel Drive
Hercules, CA 94547
antitation of Protein Samples
1-800-4BIORAD (1-800-424-6723)
Western blotting is a widely used and sensitive techniq
For Product
ue for
Technical Support:
used to calculate t
lsg_tec
he MW of
hser
an u
v_us@bio-r
nknown protei
ad.com
n based on its R
the detection and characterization of proteins. This tec
For Liter
hnique
ature Request: lsg_literature_request@bio-rad.com
f
.
In another application, a blotted dilution series of puriﬁed test
takes advantage of antibody recognition speciﬁcity tow
www
ard an
.discover.bio-r
prote
ad.com
in of known concentration is used to construct a calibration
antigenic sequence in a target protein. In a typical western
curve of signal volume versus protein amount. The curve is then
b
Molecular
lotting protocol, sol
W
ubi
eight Estimation and Quantitation of Protein Samples
lized protein is separated by SDS-PAGE
used to determine the amount of test protein in a crude sample.
and electrophoretically transferred to a nitrocellulose or PVDF
m
W
e
estern
mbran
blotting
e (Figure
is
1
a
). T
widely
he cho
used
ice of
and
dete
sensitive
ction sub
technique
strates and
for the
Chemiluminescent Western Blotting Workﬂow
v
detection
isualization
and
me
characteriza
thods can ha
tion
ve a
of
gr
proteins.
eat impa

c
This
t on t
technique
he quality
takes
o
advanta
f data fr
ge
om
of
a
antibod
western
y
b
recognition
lot.
speciﬁ city toward an antigenic
Electrophoresis of Samples
sequence in a target protein. In a typical Western blotting protocol,
Criterion
™
Gel System Precision Plus Protein
C
WesternC Standards (on Gel)
solubilized
hemilumin
protein
escent
is
de
separa
tection
ted
us
by
ing
SDS-P
a dig
AGE
ital im
and
ag
electrophoretically
ing system offers
im
transferred
portant ad
to
van
a
ta
nitrocellulose
ges over ﬁlm-
or
ba
PVDF
sed or
membrane
colorimetric
(F

igure
proced
1).
ur

e
The
s.
A
choice
comm
of
on
detection
objective
substra
of wes
tes
tern
and
blot
visualiza
experim
tion
ents
methods
is to obta
can
in
have
q
a grea
uantita
t impact on the quality of da
tive information about a tar
ta from a
get prote
W
in
estern blot.
, including its
molecular weight (MW) or concentration. For these applications,
cChemiluminescent hemiluminescent imdetection ages capusing tureda bdigital y digitaimal doging cumesystem ntation offers
Blot Transfer
p
important
resent a w
advanta
ider dyn
ges
ami
over
c ran
ﬁ
g
lm-based
e and are
or
eas
colorimetric
ier to quanti
procedures.
tate by

Criterion Blotter Precision Plus Protein
d
A
en
common
sitometry,
objective
compared
of
w
W
ith
estern
colorim
blot
etric
experiments
or ﬁlm-based
is
d
to
etec
obtain
tion.
and Membranes
WesternC Standards (on Blot)
quantitative information about a target protein, including its
Pmolecular rotein stanweight dards p(MW) lay anor intconcentraegral part tion.in a nFaor lyzithese ng wesatpplicaern bltions,otting
e
chemiluminescent
xperiments. They ca
ima
n p
ges
rovide
ca
r
ptured
eliable M
by
W s
digital
izing, an
documenta
d also serv
tion
e
a
present
s interna
a
l c
wider
ontrols
d

ynamic
for moni
range
toring
and
an el
are
ectro
easier
phores
to
is
quantita
run and
te by
e
densitometr
fﬁciency of
y
b
, compared with colorimetric or ﬁ
lot transfer. Precision Plus Prote

i
lm-based detection.
n
™
WesternC
™
standards are prestained recombinant protein markers optimized
Immunodetection of Samples
f
Protein
or direc
standards
t visualizat
play
ion in
an
ge
integral
ls, trans
part
ferr
in
ed
analyzing
blots, and
W
f
estern
or para
blotting
llel
Immun-Star WesternC StrepTactin-HRP
experiments. They can provide reliable MW sizing, and also serve as
Chemiluminescent Kit Conjugate (for Detection)
d
internal
etection
controls
in chem
for
ilu
monitoring
minescent
an
we
electrophoresis
stern blotting ap
run
plic
and
atio
efﬁ
ns
ciency
(F
of
igu
blot
re 1)
transfer
.
. Precision Plus Protein
TM
WesternC
TM
standards
T
are
he I
prestained
mmun-Star
recombinant
™
WesternC
™
protein
chemil
markers
uminesce
optimized
nt detectio
for
n k
direct
it
c
visualiza
ontains a
tion
sen
in
si
gels,
tive,

c
transferred
hemilumine
blots,
scen

t
and
subs
for
tra
parallel
te for ho
detection
rseradish
in
chemiluminescent Western blotting applications (Figure 1).
peroxidase (HRP) detection by digital imaging systems. The
Visualization of Blots
s
The
ubstr
Immun-Star
ate is speciﬁTMc

a
W
lly
esternC
designTMed

t
chemiluminescent
o generate rapid, in
detection
tense
kit
ChemiDoc XRS Imager Precision Plus Protein WesternC
li
contains
ght emiss
a
io
sensitive,
n of exten
chemiluminescent
ded duration, which
substra
is opti
te
ma
for
l for
horseradish
use
Standards (Chemiluminescent)
w
peroxidase
ith charge-
(HRP)
coup
detection
led device
by
(C
digital
CD)-b
ima
ase
ging
d ima
systems.
gers, su
The
ch a
substra
s the
te
Mis ospeciﬁlecula rcally Imagdesigned er
®
Chemto iDgeneraoc
™
Xte RSra impid,ag iintense ng systelight m. emission of
extended duration, which is optimal for use with charge-coupled
T
device
he Prec
(CCD)-based
ision Plus Pro
ima
tein
gers,
Wes

te
such
rnC st
as
and
the
ards,
Molecular
StrepTactin
Ima
-HR
ger
P ®

c
ChemiDoc
onjugate,
TM
Im
XRS ima
mun-Sta
ging system.
r WesternC

chemiluminescent detection
MW Estimation or Quantitation
kit, and the ChemiDoc XRS imager can be used to estimate the
MThe W aPrecision nd amounPlus t (ng)Protein of proteWinesternC samplestandards,s in wester nStrepT blottiactin-HRP ng
FiFgig. . 11. . WWesetseternrn blbotlottitningg wwoorkrkﬂfoloww frforomm SSDDSS-P-APAGGEE tot o vivsiusaulaizliazatitoionn aanndd aannaalylsysisis..
e
conjuga
xperime
te,
nt

s
Immun-Star
. Blotted Wes
W
te
esternC
rnC stan
chemiluminescent
dards are used to
detection
graph log
kit,
T
T
h
h
e i
e
n
in
vo
vo
lv
l
e
v
m
em
en
e
t o
nt
f P
of
r
P
e
r
c
e
is
c
i
i
o
s
n P
ion
lu
P
s P
lus
ro
P
t
r
e
o
in W
tein
e
W
st
es
rn
te
C s
rnC
tan
s
d
ta
a
n
r
d
s a
ard
t e
s at
ch s
eac
te
h
p i
st
n
ep
producing a chemiluminescent western blot is shown on the right. Key equipment
M
and
W v
the
ersu
ChemiDoc
s relative m
XRS
igra
ima
tion
ger
dist
can
ance
be
(R
used to estimate the MW
in producing a chemiluminescent Western blot is shown on the right. Key
and amount (ng) of protein samples in Western
f
). The plot can then be and reagents are shown on the left.
blotting experiments.
equipment and reagents are shown on the left.
Blotted WesternC standards are used to graph log MW versus relative
migration distance (R
f
). The plot can then be used to calculate the
MW of an unknown protein based on its R
f
. In another application, a
blotted dilution series of puriﬁ ed test protein of known concentration
is used to construct a calibration curve of signal volume versus
protein amount. The curve is then used to determine the amount of
test protein in a crude sample. StrepTactin is a trademark of Institut für Bioanalytick GmbH.
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 45
BioRad membrane Protocol.indd 45 10/26/07 11:43:21 AM
Page 1 | Page 2 | Page 3 | Page 4 | Page 5 | Page 6 | Page 7 | Page 8 | Page 9 | Page 10 | Page 11 | Page 12 | Page 13 | Page 14 | Page 15 | Page 16 | Page 17 | Page 18 | Page 19 | Page 20 | Page 21 | Page 22 | Page 23 | Page 24 | Page 25 | Page 26 | Page 27 | Page 28 | Page 29 | Page 30 | Page 31 | Page 32 | Page 33 | Page 34 | Page 35 | Page 36 | Page 37 | Page 38 | Page 39 | Page 40 | Page 41 | Page 42 | Page 43 | Page 44 | Page 45 | Page 46 | Page 47 | Page 48 | Page 49 | Page 50 | Page 51 | Page 52 | Page 53 | Page 54 | Page 55 | Page 56 | Page 57 | Page 58 | Page 59 | Page 60 | Page 61 | Page 62 | Page 63 | Page 64 | Page 65 | Page 66 | Page 67 | Page 68 | Page 69 | Page 70 | Page 71 | Page 72 | Page 73 | Page 74 | Page 75 | Page 76