A New Dry Blotting System for Rapid Protein Transfer ... - Invitrogen

A New Dry Blotting System for Rapid
Protein Transfer from Polyacrylamide
Gels to Membranes
Tom Diller 1 , Jennifer Miller 1 , Tim Updyke 1 , Galia Feldman 2 ,
Inbal Simcha 2 , and Cory Szafranski 1
1 Invitrogen Corporation, Carlsbad, CA, USA
2 Invitrogen Corporation, Ness Ziona, Israel
Introduction
Western blotting is a standard method for the immunologic
visualization of proteins that have been separated by gel
electrophoresis. Many techniques are available for transferring
(blotting) proteins from gels to membranes prior to
immunodetection, including electroblotting gels onto capture
membranes via “wet transfer” techniques. These wet
techniques are typically performed in a tank with a large
volume of buffer and require one to two hours or more for setup
and transfer of proteins. Semi-dry protein transfer
alleviates some of these drawbacks but requires significant
effort to “sandwich” together the layers of gel, blotting paper,
and membranes, and semi-dry blotting still requires about an
hour to transfer the proteins. In this study, we evaluate a new
“dry blotting” device, called the iBlot Dry Blotting System, for
protein transfer after gel electrophoresis. This buffer-free
system is equipped with its own integrated power supply,
utilizes preassembled transfer stacks with an integrated
nitrocellulose membrane, and transfers proteins in a very short
period of time (six to seven minutes). We compare dry
blotting to conventional wet and semi-dry blotting for different
protein standards and E. coli or Human colon cancer cell
lysates at various load levels.
Experimental Section
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was performed with samples using four distinct
types of gradient mini gels:
NuPAGE ® Novex ® 4-12% Bis-Tris Gels with MES running
buffer (Invitrogen)
Novex ® 4-20% Tris-Glycine Gels (Invitrogen)
5-20% Tris-Glycine Gels (Brand X)
4-20% Tris-Glycine Gels (Brand Y)
4-12% Bis-Tris Gels (Brand Z)
To compare dry blotting to wet blotting, gels were loaded with
three samples of varying type and protein mass amounts,
listed by lane number below (for Figures 1-4*):
1. 3 µl SeeBlue ® Plus2 Pre-Stained Protein Standard
(Invitrogen)
2. 0.5 µl MagicMark XP Western Protein Standard
(Invitrogen)
3. 1.0 µl MagicMark XP Western Protein Standard
4. 2.0 µl MagicMark XP Western Protein Standard
5. 3.0 µl MagicMark XP Western Protein Standard
6. 10 ng E. coli lysate
7. 25 ng E. coli lysate
8. 50 ng E. coli lysate
9. 100 ng E. coli lysate
10. 200 ng E. coli lysate
11. 300 ng E. coli lysate
12. 6 µl SeeBlue ® Plus2 Pre-Stained Protein Standard
(*Note: Figure 4 contains an extra lane of 3 µl SeeBlue ® Plus2
Pre-Stained Protein Standard between lanes 5 and 6).
The following samples were loaded onto a 4-12% Bis-Tris gel
(Brand Z) to compare dry blotting to semi-dry blotting (Figure
5), by lane number:
1. 0.0625 µg SW480 Human colon cancer cell lysate
(Alexis) (control)
2. 0.125 µg SW480 lysate
3. 0.25 µg SW480 lysate
4. 0.5 µg SW480 lysate
5. 1.0 µg SW480 lysate
6. 2.0 µg SW480 lysate
7. 5 µl SeeBlue ® Plus2 Pre-Stained Protein Standard
8. 0.5 µl MagicMark XP Western Protein Standard
9. 1.0 µl MagicMark XP Western Protein Standard
10. 2.0 µl MagicMark XP Western Protein Standard
11. 4.0 µl MagicMark XP Western Protein Standard
12. 5 µl SeeBlue ® Plus2 Pre-Stained Protein Standard
SDS-PAGE protocols and protein transfers to nitrocellulose
membranes were performed according to manufacturers’
instructions. Dry versus wet transfers were compared using
the iBlot Dry Blotting device and an Invitrogen XCell II
(Wet) Blot Module (Figures 1-4). Immunodetection of proteins
on the resulting membranes in Figures 1-4 was performed
using a WesternBreeze ® Chromogenic Immunodetection Kit
(Invitrogen) for detection of rabbit primary antibodies. An
anti-E. coli primary antibody (DAKO) was used (1:2000) with a
chromogen development time of 15 minutes.
A semi-dry transfer apparatus (Trans-Blot SD, Bio-Rad) was
used to compare iBlot dry transfer to a semi-dry transfer in
Figure 5. Immunodetection for Figure 5 was performed with a
WesternBreeze ® Chromogenic Immunodetection kit for
detection of mouse primary antibodies. Anti-Actin (1:5,000)
and anti-Tubulin (1:10,000, both Sigma) and a chromogen
development time of 15 minutes were used for detection in
this case.
Results
Figure 1 compares the resulting protein blots after transfer
with the iBlot Dry Blotting System (6-minute, 23 volt
transfer), and wet transfer (60 minutes at 30 volts) for protein
mixtures separated with NuPAGE ® Novex ® gels. Upon close
visual inspection, the blots appear very similar, not only in the
quality of the blot (band and lane shape) but also in the
pattern of transferred proteins, the ability to detect proteins at
the lower loading amounts, and, thus, sensitivity.
Dry transfer Wet transfer
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Figure 1. Membranes from NuPAGE ® Novex ® 4-12%
Bis-Tris Gels with NuPAGE ® MES Running Buffer.
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Figure 2 compares data for dry blotting the same samples from
Novex ® Tris-Glycine Gels for 6 minutes at 23 volts with data
obtained for wet blotting with the protocol-recommended 60
minutes at 25 volts. Again, the dry transfer membrane data is
visually comparable to the wet transfer data. Note that protein
bands for the pre-stained SeeBlue ® Plus2 Standard (lanes 1
and 12) are more evident on the dry blotted membrane than
with wet blot, particularly in the higher molecular weight
region. This may indicate that the iBlot TM transfer system
possesses greater sensitivity as compared to wet transfer of
some protein samples and gel types.
Dry transfer Wet transfer
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Figure 2. Membranes from Novex ® 4-20% Tris-Glycine
Gels.
Figures 3 and 4 are similar comparisons of the same samples
transferred from other brands of Tris-Glycine gels to
nitrocellulose membranes (6 minutes/23 volts with dry
transfer; 60 minutes/25 volts for Brand X and Brand Y gels via
wet transfer). The data indicate that dry transfer of proteins
using the iBlot Dry Blotting System yields results that are
very comparable to results obtained using standard wet
transfer methods that have been used historically for protein
transfer and which take significantly longer to perform. The
quality of the data and the sensitivity of the western detection
technique seem reduced in the data obtained with Brand X and
Y gels as compared to that obtained from NuPAGE ® and
Novex ® gels, while the blots from NuPAGE ® Novex ® Bis-Tris
gels appear to outperform the other gel types with respect to
band quality and sensitivity.
Dry transfer Wet transfer
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Figure 3. Membranes from 5-20% Tris-Glycine Gels
(Brand X).
Dry transfer Wet transfer
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Figure 4. Membranes from 4-20% Tris-Glycine Gels
(Brand Y).
Dry transfer of proteins (6 minutes/23 volts) from a 4-12%
Bis-Tris gel to nitrocellulose is compared with semi-dry (60
minutes/20 volts) protein transfer in Figure 5. Data for these
gels, as well as NuPAGE ® Novex ® gels and other competitive
gels we compared (data not shown), indicate that dry transfer
of proteins using the iBlot dry blotting system is superior to
semi-dry blotting for detection of proteins. The ability to
detect tubulin and actin at lower protein masses in lanes 1-6
and the more pronounced bands evident in the MagicMark
XP Standard lanes (8-11) of Figure 5 demonstrate this higher
sensitivity.
Dry transfer Semi-dry
transfer
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Figure 5. Membranes from 4-12% Bis-Tris Gels (Brand
Z).
Summary
For the conditions and samples tested, the iBlot Dry Blotting
System provides overall visual immunodetection results that
are comparable to results obtained using a standard wet
blotting system but in a fraction of the time and with both less
effort and less volume of reagents. When used with the
iBlot system, visual immunodetection results of Brand X and
Y Tris-Glycine gels are similar to results obtained using Novex ®
Tris-Glycine gels, indicating that the dry blotting system can be
used with gels from a variety of sources. In addition, dry
protein transfer enables significantly more sensitive detection
of proteins than semi-dry protein transfer and requires much
less time.
Finally, for dry versus wet protein transfer, the quality and
sensitivity of the resulting protein blots are comparable among
all the gels included within this study, yet the best results
appear to be obtained from NuPAGE ® Novex ® gels. These
results indicate that western blot protein transfers can be
optimized by choosing the blotting procedure and gel type that
is best suited to your sample and that satisfactory results can
now be obtained with dry blotting in just minutes as opposed
to waiting for hours for data from the laboratory.
For more information, visit www.invitrogen.com/iBlot.
Invitrogen Corporation
1600 Faraday Avenue, Carlsbad, CA 92008 USA
Contact: protein.analysis@invitrogen.com
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