Glycine reprobing protocol using the iBlot® 7

APPLICATION NOTE
iBlot® 7-Minute Blot
Glycine reprobing protocol using the
iBlot® 7-Minute Blot
Introduction
Western blotting is a standard method for immunologic
visualization of proteins that have been separated by
gel electrophoresis. When multiple proteins need to be
detected on a single membrane, the traditional method
used involves stripping the primary and secondary
antibodies from the blot and reprobing with different
primary and secondary antibodies.
One common method of antibody removal is the use
of a heated, low-pH solution containing glycine. The
combination of the heated buffer with the low pH causes
dissociation of the antibody-antigen complex.
In this application note, we compare reprobing with
glycine after dry blotting of an E. coli lysate at various
protein quantities with the iBlot® 7-Minute Blot to
reprobing the same samples following conventional wet
blotting.
Methods
Prepare stripping buffer
Safety—please note:
The glycine solution is pH 2. This protocol should be
performed with proper safety precautions, including the
use of protective eyewear and thermal gloves. Be especially
careful when microwaving a low pH solution.
Prepare 0.1 M glycine pH 2:
1. Create a stock solution of 500 mM glycine. Dissolve
9.38 g of glycine in 250 mL distilled deionized water
(DDW), then add concentrated HCl drops until a pH of 2
is reached as measured with a pH meter.
2.To prepare 100 mM (0.1 M) glycine, dilute the 500 mM
stock solution with DDW. The amount of DDW added
will depend on the amount of fresh stripping buffer
needed (e.g., to make 400 mL of stripping buffer, add
80 mL of the stock solution and 320 mL of DDW).
Stripping protocol (for one mini membrane)
1. Microwave the 0.1 M glycine solution until it boils.
2.Using tweezers, place the membrane in the 0.1 M
glycine solution for 7 min. Repeat this incubation
3 times, each time using a fresh glycine solution.
3.Wash the membrane with phosphate buffered saline
with 0.05% Tween 20 (PBST) for 10 min. Repeat this
step twice, using fresh PBST solution each time.
After washing, continue your western protocol for
reprobing as usual.
Validation process
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was performed with
samples using Novex® 4–12% BisTris Gels with MES running buffer.
To compare stripping and reprobing
after dry blotting and wet blotting,
gels were loaded with E. coli lysate of
varying protein mass amounts, listed
by lane number from left to right
(Figures 1–2).
To further verify that stripping of
the first antibody complex took
place, a duplicate membrane was
stripped and then incubated with a
secondary antibody. Any remnants of
the primary antibody observed here
dicate incomplete transfer (Figures
3-4).
SDS-PAGE protocols and protein
transfers to nitrocellulose (NC) and
polyvinylidene difluoride (PVDF)
membranes were performed according to manufacturers’ instructions.
Dry and wet transfers were done
using the iBlot® 7-Minute Blot and an
XCell II™ Blot Module (wet blotting)
(Figures 1–4). Immunodetection of
proteins on the resulting membranes
in Figures 1–4 was performed using
an
anti-E. coli primary antibody (1:2,000)
and a secondary goat anti-rabbit
HRP antibody (1:10,000). For detection, the membranes were developed
with the ECL™ Western Blotting
System (Amersham) with a development time of 1 min.
Results
Figure 1 compares the resulting
E. coli protein blots transferred with
the iBlot® 7-Minute Blot (program P3
for 7 min) and by wet transfer
(60 min at 30 V) for protein mixtures
separated with NuPAGE® gels
before and after stripping and
reprobing on a NC membrane. Upon
comparison, the results before
and after stripping and reprobing
appear very similar in the ability to
detect proteins at the lower loading
amounts. Sensitivity is thereby
sustained between dry and wet
blotting.
Figure 2 compares the resulting
E. coli protein blots transferred with
the iBlot® 7-Minute Blot (program P3
for 7 min), and wet transfer (60 min
at 30 V) for protein mixtures separated with NuPAGE® gels before and
after stripping and reprobing on a
PVDF membrane. Upon comparison,
the results before and after stripping
and reprobing appear very similar in
the ability to detect proteins at the
lower loading amounts. Sensitivity is
thereby sustained between dry and
wet blotting, as also observed with
NC membranes.
Figures 3 and 4 are the same resulting E. coli protein blots transferred
with the iBlot® 7-Minute Blot and
wet transfer after stripping (from
Figure 2) and incubated with the
secondary antibody alone to validate
stripping efficiency. No bands were
detected and stripping efficiency was
confirmed.
Summary
Stripping and reprobing with 0.1 M of
glycine at pH 2 was performed on NC
and PVDF membranes containing
E. coli protein lysate transferred with
the iBlot® 7-Minute Blot and the wet
blotting system. The results obtained
after stripping and reprobing of a
membrane after dry blotting with the
iBlot® 7-Minute Blot are comparable
to the results received with the
wet blotting system. Not only is the
protein blotted in just a few minutes
with the iBlot® 7-Minute Blot,
compared with 60–120 min with wet
blotting, but the quality of the results
after stripping and reprobing is
reproducible and similar to the first
probing. This indicates that standard
western analyses can benefit from
dry blotting with significant time
savings without compromising data
quality.
Before stripping and reprobing
1
2
3
Dry transfer
4
After stripping with 0.1 M glycine
pH 2 and reprobing with primary and
secondary antibodies
5
Wet transfer
Dry transfer
Wet transfer
Figure 1. NC membranes from NuPAGE® Novex® 4–12% Bis-Tris Gels with NuPAGE® MES Running Buffer before and after stripping and
reprobing with 0.1 M glycine pH 2. Gels were loaded with E. coli lysate of varying protein mass amounts, from left to right: Lane 1. 78 ng
E. coli lysate; Lane 2. 156 ng E. coli lysate; Lane 3. 312 ng E. coli lysate; Lane 4. 625 ng E. coli lysate; and Lane 5. 1.25 μg E. coli lysate.
Before stripping and reprobing
Dry transfer
After stripping with 0.1 M glycine
pH 2 and reprobing with primary and
secondary antibodies
Wet transfer
Dry transfer
Wet transfer
Figure 2. PVDF membranes from NuPAGE Novex 4–12% Bis-Tris Gels with NuPAGE MES Running Buffer before and after stripping
and reprobing with 0.1 M glycine pH 2. Gels were loaded with E. coli lysate of varying protein mass amounts, from left to right: Lane 1. 78
ng E. coli lysate; Lane 2. 156 ng E. coli lysate; Lane 3. 312 ng E. coli lysate; Lane 4. 625 ng E. coli lysate; and Lane 5. 1.25 μg E. coli lysate.
®
®
®
After dry blotting, stripping, and
reprobing with secondary antibody only
After wet blotting, stripping, and
reprobing with secondary antibody only
NC
NC
PVDF
Figure 3. NC and PVDF membranes from NuPAGE® Novex®
4–12% Bis-Tris gels with NuPAGE® MES Running Buffer
transferred with the iBlot® 7-Minute Blot. Gels were loaded
with E. coli lysate of varying protein mass amounts, from
left to right: Lane 1. 78 ng E. coli lysate; Lane 2. 156 ng E.
coli lysate; Lane 3. 312 ng E. coli lysate; Lane 4. 625 ng E.
coli lysate; and Lane 5. 1.25 μg E. coli lysate.
PVDF
Figure 4. NC and PVDF membranes from NuPAGE® Novex®
4–12% Bis-Tris gels with NuPAGE® MES Running Buffer
transferred with the wet system. Gels were loaded with
E. coli lysate of varying protein mass amounts, from left
to right: Lane 1. 78 ng E. coli lysate; Lane 2. 156 ng E. coli
lysate; Lane 3. 312 ng E. coli lysate; Lane 4. 625 ng E. coli
lysate; and Lane 5. 1.25 μg E. coli lysate.
For more information, go to lifetechnologies.com/iblot
Ordering information
Product
Quantity
Cat. No.
NuPAGE Novex 4–12% Bis-Tris Gel 1.0 mM, 12 well
Box of 10
NP0322BOX
NuPAGE MES Running Buffer 20X
500 mL
NP0002
iBlot® Gel Transfer Device
1 unit
IB1001
iBlot Transfer Stack, Regular (nitrocellulose)
10 sets/box
IB301001
iBlot Transfer Stack, Mini (nitrocellulose)
10 sets/box
IB301002
iBlot® Transfer Stack, PVDF Regular
10 sets/box
IB401001
®
®
®
®
®
iBlot Transfer Stack, PVDF Mini
10 sets/box
IB401002
XCell II™ Blot Module
1 unit
EI9051
Nitrocellulose membranes, 0.2 µm for mini gels
20/box
LC2000
®
Nitrocellulose membranes, 0.45 µm for mini gels
20/box
LC2001
PVDF membranes, 0.2 µm for mini gels
20/box
LC2002
Invitrolon™ PVDF, 0.45 µm for mini gels
20/box
LC2005
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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registered trademark of Uniquema Americas LLC. ECL is a trademark of GE Healthcare.
Printed in the USA. CO01679 0112
lifetechnologies.com

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