Convenient, reproducible, and automated blot processing and gel

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Convenient, reproducible, and automated blot
processing and gel staining using Processor Plus
L. Hennessy, N. Laird, and W. Stochaj
Amersham Biosciences, San Francisco, CA
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and protein-nucleic acid interactions as well. Although
conceptually simple, the actual processing of the
protein blot to detect molecules of interest is a tedious
and labour-intensive task, involving successive incubations with a series of reagents and washes. Likewise,
gel staining (particularly silver staining) involves
numerous incubations with stains and rinses. To
reduce the “hands-on” time requirements for Western
blotting and gel staining and to improve the reproducibility of these procedures, Amersham Biosciences
has developed an instrument that automates
the time-consuming steps of these routine protocols.
Hoefer™ Processor Plus™ automates membrane
processing and gel staining following polyacrylamide gel
electrophoresis. Ten protocols are pre-programmed for
use with Western detection and gel staining kits from
Amersham Biosciences. Users can also program
their own protocols or modify the standard protocols.
Protein blot processing and silver staining with this
instrument is convenient, reproducible, and sensitive.
Introduction
Transfer of proteins from polyacrylamide gels to an
immobilizing matrix, commonly referred to as Western blotting, is a widely used technique (1). Immobilization of proteins on a microporous membrane
preserves the resolution of the electrophoretic separation and permits further analysis using a broad array
of techniques, in essence providing a “third dimension” to protein characterization. While the most
commonly used method is the indirect detection of
protein antigens with secondary antibodies, this
technology has been extended to examine other
types of protein-protein, protein-carbohydrate,
Automated processing
Hoefer Processor Plus automates fluid delivery and
timing steps for both membrane processing and polyacrylamide gel staining (Fig 1). Processor Plus can
accommodate up to four mini-blots (8 × 7–9.5 cm) or
two standard-size blots (14–16 × 16 cm) for membrane
processing. For silver and commassie staining, the
instrument handles 1–6 mini-gels or 1–2 standard-size
gels. It utilizes a rocking tray with a unique rhomboid
shape to produce rapid and thorough mixing when
the tray is rocked at a shallow angle. This minimizes
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Fig 1. Features of the Processor Plus Instrument. (A) Instrument panel—10 membrane keys are used in operating and programming the instrument.
A 32-character LCD guides the user through set-up and programming, and indicates instrument status during processing; (B) Protocol key memory
chip stores a single protocol. Protocols can be written onto the protocol key from the instrument or copied from the protocol key into instrument
memory; (C) Blot processing lid; (D) Blot processing tray; (E) Gel staining tray; (F) Magnets to hold gel in place.
Life Science News 3, 1999 Amersham Biosciences
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are used, and avoids the accidental alteration of
protocols by another user. Additional keys are
available separately.
reagent usage without sacrificing thorough washing
and gentle treatment of blots and gels.
A microprocessor controls the rocker motor for the
tray, the pumps, and the 10-port valve, allowing
membrane and staining protocols to be programmed
by the user so the instrument may be left unattended.
Fluid is delivered by one of two peristaltic pumps. For
blot processing, reagent is supplied through a 10-port
valve to a four-port manifold on the blot processing
lid. Valves allow from one to four chambers to be
used. By tilting the tray backwards, reagents are
drained from each tray chamber to a common collection point, and are removed by a second isolated
pump. All reagents are collected in a common waste
container. For gel staining, reagents are supplied to
and removed from the tray through the 10-port valve
using a single pump head. This allows hazardous
wastes to be segregated for proper disposal.
Blotting results
A comparison of automated and manual blot processing was performed (Fig 2). Equal amounts of bovine
serum albumin (BSA) were filtered onto Hybond™
-C
membrane (RPN1210C) using Hoefer PR 648 Slot
Blot apparatus (80-6095-58). Blots were processed
with anti-BSA primary antibody either manually or
with Processor Plus. Immunodetection was performed
with the chromogenic alkaline phosphatase substrate
BCIP/NBT. The resulting blots were scanned. The
calculated average volumes (area × density) for slots in
the two membranes were essentially indistinguishable
from one another (7.6 ± 0.26 Processor Plus compared
with 7.5 ± 0.32 manual).
Processor Plus
The instrument is delivered preprogrammed with four
membrane and eight gel staining protocols that have
been developed for use with Amersham Biosciences
Biotech blotting and staining kits. In addition, up
to 10 custom protocols may be stored—five for
membrane processing and five for gel staining. Each
protocol may contain up to 30 steps. The keypad on
the front panel facilitates easy programming and
editing, allowing quick addition or deletion of steps.
Additional features that can be programmed into any
step in a protocol include a “hold” (which prevents
the instrument from proceeding to the next step until
it is advanced manually) and a “beep” (which sounds
an audible alarm at the end of a step). These features
are useful when the user may want to run the unit
overnight but be present to monitor a critical step
the next morning.
manual
Fig 2. Comparison of automated and manual blot processing results.
Identical amounts of BSA (25 ng) were immobilized on Hybond-C
membrane using Hoefer PR 648 Slot Blot apparatus. Blots were
incubated first with rabbit anti-BSA primary antibody (1:125 000),
then with biotinylated goat anti-rabbit secondary antibody (1:2 500),
and finally with streptavidin-alkaline phosphatase (1:2 500). Colour
development was performed with chromogenic alkaline phosphatase
substrate BCIP/NBT. The resulting blots were scanned in transmittance mode using a Sharp JX-330 scanner and analysed using
ImageMaster™ VDS software.
A serial printer can be connected to Processor Plus for
report generation. The report provides information on
each step including the volume of reagents used, the
time (minutes) for each step, and a validation report
form, with spaces for sample ID, date, and operator.
As an added benefit, the required volume of each
reagent for a specific protocol can also be printed
using the set-up menu.
To demonstrate day-to-day reproducibility, two-fold
serial dilutions of immuno-isolated human Apo-AI
containing lipoproteins were separated on 12%
polyacrylamide gels (containing Tris/glycine/SDS)
and transferred to Hybond ECL™ nitrocellulose
(RPN68D). Three blots were processed on consecutive days. The blots were processed using ECL Plus™
Chemiluminescent Detection Kit (RPN2132).
The films were scanned as described for Figure 2.
The absorbance data for the blots were nearly
identical (Fig 3).
A novel feature is the separate protocol key that
contains a memory chip with the capacity to store
one membrane or staining protocol. The protocol key
allows critical protocols to be stored away from the
instrument. After inserting the key into the key slot,
the protocol on the key can be transferred to or from
the Processor Plus memory. This is particularly useful
in multi-user environments where several protocols
Life Science News 3, 1999 Amersham Biosciences
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albumin
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transferrin
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albumin +
transferrin
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no primary
antibody
day 1
day 2
Fig. 4. Chamber-to-chamber containment. Thirty micrograms of
immuno-isolated human Apo-AI containing lipoproteins were separated on a 12% polyacrylamide gel containing Tris/glycine/SDS, and
transferred to Hybond ECL membrane. The blot was cut into strips
and four of the strips were simultaneously processed in the mini-blot
processing tray. Each chamber contained a different solution of primary antibody: 1) goat anti-albumin (1:200 000); 2) goat anti-transferrin (1:200); 3) goat anti-albumin (1:200 000) plus goat anti-transferrin
(1:200); or 4) blocking solution without primary antibody. After washing, all blots were incubated with the secondary antibody (mouse antigoat IgG horseradish peroxidase conjugate, 1:10 000). The blots were
detected with ECL Plus Chemiluminescent Detection Kit and exposed
to film for 5 min. Exposure of the blot containing no antibody was
extended to 30 min to check for contamination.
day 3
2.5
day 1
2.0
O.D.
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1.5
day 2
day 3
1.0
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0
0.07
0.13
0.20
0.26
ECL Plus Chemiluminescent Detection Kit. As shown
in Figure 4, there was no cross-contamination
observed between the tray chambers. A duplicate
experiment generated identical results. The results
demonstrate that the tray design prevents
chamber-to-chamber contamination.
0.34
Rf
Fig 3. Day-to-day reproducibility. Two-fold serial dilutions of immunoisolated human Apo-AI containing lipoproteins were separated on 12%
polyacrylamide gels containing Tris/glycine/SDS and transferred to
Hybond ECL membrane. The blots were processed on consecutive
days. The blots were incubated first with goat anti-transferrin primary
antibody (1: 200), then with mouse anti-goat IgG horseradish peroxidase conjugate (1:10 000). Chemiluminescent detection was performed using ECL Plus Chemiluminescent Detection Kit. All blots were
exposed to film for 7.5 min and then scanned as described in Figure
2. Lipoprotein samples were generously provided by Dr. Clive
Pullinger, UC San Francisco, Cardiovascular Research Institute.
The versatility of Processor Plus is shown by the
blots and gels in Figure 5. The instrument was used
to process a mini-Western blot, a silver-stained DNA
gel, a 2-D blot, and a silver-stained 2-D gel. Both blot
processing and gel staining can be easily performed
using Processor Plus.
To demonstrate the absence of reagent exchange
(cross-contamination) between the four chambers in
the mini-blot processing tray, strip blots were prepared
that contained 30 µg of immuno-isolated human ApoAI containing lipoproteins. One strip was placed into
each chamber of the mini-processing tray. After blocking, different primary antibody solutions were added
to each chamber as follows: 1) anti-human albumin,
2) anti-human transferrin, 3) both anti-albumin and
anti-transferrin or 4) blocking solution without primary antibody. The blots were incubated with the
secondary antibody. Detection was performed using
Summary
Hoefer Processor Plus makes Western blotting and
gel staining simple, convenient, and reproducible.
Amersham Biosciences ECL and ECL Plus
Chemiluminescent Detection Kits and PlusOne™
Silver Staining Kits can be used for additional
convenience and high quality results.
References
1. Towbin, H. et al., Proc. Natl. Acad. Sci. USA 76,
4350–4354 (1979).
Life Science News 3, 1999 Amersham Biosciences
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Fig 5. Examples of Western blot processing and gel staining using
Processor Plus.
A) Decreasing amounts of TCA/acetone-precipitated E. coli extracts
were separated on a 12% Tris/glycine/SDS gel, transferred to Hybond-P
membrane (RPN2020F) and then processed in Processor Plus using
the ECL Plus protocol. The blot was incubated first with rabbit antiGroEL primary antibody (1:600 000), then with biotinylated goat antirabbit secondary antibody (1:4 000), and finally with streptavidin
horseradish peroxidase congugate (1:4 000). B) PCR amplicons
analysed for single-stranded conformation polymorphisms (SSCP)
stained with PlusOne DNA Silver Staining Kit (17-6000-30), using
silver staining protocol 1 of Processor Plus. C) Immunoblot of cell culture extract of gliobastoma multiforme brain tumour cells. The blot was
incubated with mouse anti-p53 primary antibody (1:30 000) and then
goat anti-mouse horseradish peroxidase conjugate (1:5 000). Immunodetection was performed with ECL Chemiluminescent Detection Kit
(RPN2108). The human cell culture extract and monoclonal antibody
were generously provided by Dr. Mike Harrington, Huntington Medical
Research Institute, Pasadena, CA. D) 2-D gel of a cell culture extract of
gliobastoma multiforme brain tumour cells stained with PlusOne Silver
Staining Kit, Protein (17-1150-01), using silver staining protocol 2 of
Processor Plus. Proteins detected on the 2-D blot are boxed in yellow.
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ORDERING INFORMATION
Processor Plus - Blot Processing and Gel Staining System
Processor Plus Instrument
1
80-6444-04
1
80-6444-23
Base Unit, Reagent Tubing, Protocol Key
Tray Options
Blot Processing Tray Pack
Complete with Tray Base, Disposable Mini and
Standard Tray, Lid, Reagent Bottles and Rack
Staining Tray Pack 19 × 29 cm
1
80-6444-80
Complete with Gel Staining Tray Base, PTFE-coated Tray, and Lid
Staining Tray Pack 29 × 35 cm
1
80-6445-18
Complete with Gel Staining Tray Base, PTFE-coated Tray, and Lid
Life Science News 3, 1999 Amersham Biosciences
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