GST96detnr11.

qxd (rev) 8/12/98 1:05 pm Page 1

I N N O V A T I O N S F O R U M

Quantitative detection of GST fusion proteins in

bacterial lysates using a 96-well array
P. A. Bell, R. W. Dunst and D. Garvin
Amersham Biosciences, Piscataway, NJ and Milwaukee, WI USA

The GST 96-Well Detection Module uses immobilized 96-Well Detection Plate. After incubation for 1 hour
R polyclonal anti-GST antibody for capture of GST fusion to allow binding of rGST Protein to the immobilized
E proteins. Captured proteins are detected using a Anti-GST Antibody, each well was washed thoroughly
C horseradish peroxidase/anti-GST conjugated antibody. with Wash Buffer (1× PBS/0.05% Tween 20). Captured
O E The system can detect as little as 1 ng of recombinant rGST Protein was detected by a 1 hour incubation with
M X a 1:1000 dilution of HRP/Anti-GST Conjugate, thor-
GST. That sensitivity is 10 to 100 times greater than
B P
capture plates which use immobilized glutathione. ough washing and addition of the soluble horseradish
I R
The capture of GST fusion proteins is very specific, peroxidase substrate, 3,3',5,5'-tetramethylbenzidine
N E
A S and the pre-blocked plates exhibit low, non-specific (TMB). The absorbance of each well at 450 nm was
N S background binding even from complex protein mixtures determined using a 96-well plate reader.
T I such as bacterial sonicates. Screening of as many as
Figure 1 shows the absorbance curve obtained from
O 96 samples per plate can be rapidly performed with
samples containing from 10 pg to 10 µg of recombi-
P N the GST 96-Well Detection Module.
nant GST per well. A reproducibly strong signal
R
O Introduction above background is obtained for as little as 1 ng of
T rGST Protein. The assay appears to be linear over
Genetic fusion of various genes to purification tags
E two logs of captured target protein concentration.
has been exploited to facilitate the purification and
I It should be noted that assays of GST fusion proteins
detection of a variety of biologically important pro-
N with different fusion partners may not exhibit the
teins (1). A rapid, specific means to assay the pres-
same sensitivity as that obtained with rGST.
ence and amount of fusion protein expressed is
required for high-throughput screening of bacterial
lysates and other samples. For glutathione S-trans-
ferase (GST) fusion proteins (2), the GST 96-Well 1.50
Detection Module provides the means for accurate,
high-throughput screening.

Polyclonal Anti-GST Antibody immobilized in the

wells of a 96-well plate permits the strong, specific
capture of all GST species in a complex mixture.
A450

Pre-blocked wells prevent non-specific binding of 0.75

non-GST proteins. After unbound proteins are
washed from the wells, captured GST fusion proteins
can be detected using the HRP/Anti-GST Conjugate
provided in the GST 96-Well Detection Module.
Alternatively, an antibody directed against the fusion
partner allows a specific GST fusion protein to be
detected. The amount of immobilized protein can
0
be quantitated by including a dilution series of the
0.01 0.1 1 10 100 1,000 10,000
supplied recombinant GST (rGST). ng rGST/well

Standard plate well GST detection

A serial dilution of rGST Protein was prepared in
1× Blocking Buffer (3% nonfat dry milk in 1× PBS/0.05%
Tween™ 20). Aliquots (100 µl) of each appropriate
dilution were applied directly into the wells of a GST
Figure 1. Detection of Recombinant GST with the GST 96-Well
Detection Module. The indicated amounts of rGST Protein were applied
to the wells of a GST 96-Well Capture Plate and detected by the stan-
dard protocol using HRP/Anti-GST Conjugate described in the text.
Detection was performed using TMB substrate, and absorbance of each
well was read at 450 nm in a 96-well plate reader.

Life Science News 1, 1998 Amersham Biosciences

1
GST96detnr11.qxd (rev) 8/12/98 1:05 pm Page 2

,
I N N O V A T I O N S F O R U M


,



antibodies directed against the fusion partner, the fol-
1.5
Amersham Biosciences lowing experiment was performed. A luciferase gene
fragment was inserted into the multiple cloning site

,

,



of the GST gene fusion vector pGEX-6P-1. Sixty-four
1.2 Vendor Y
randomly selected ampicillin resistant transformant
colonies of E. coli BL21 were used to inoculate 3 ml
Vendor X of 2× YTA and grown overnight at 37 °C. Expression
0.9 was induced by addition of IPTG to a final concen-

,,



,



A450

tration of 0.1 mM and continued culture growth for

2 hours at 37° C. Lysates of 1.5 ml aliquots of each
0.6 induced culture were prepared by sonication.

Aliquots (50 µl) of each cleared sonicate were mixed

0.3
0.0
0.1 1 10 100
ng rGST
Figure 2. Comparison of GST Capture Plates from various vendors.
The indicated amounts of rGST Protein were applied to the wells of
GST Capture plates from the indicated vendors and detected by the
standard protocol using HRP/Anti-GST Conjugate described in the
text. Detection was performed using TMB substrate and absorbance
of each well was read at 450 nm in a 96-well plate reader.
Comparison of GST detection plates
GST fusion proteins are captured in the GST 96-Well
Detection Module using immobilized Anti-GST Anti-
body in the plate wells. GST capture plates from sev-
eral other vendors utilize immobilized glutathione for
capture. The GST 96-Well Detection Module was
compared with plates from Vendor X and Vendor Y
with an equal volume of 2× Blocking Buffer and
applied directly to the wells of a GST 96-Well
Detection Plate. Three identical sets of serial dilutions
of rGST were applied into the wells of three rows of
the same plate. After allowing samples to bind for
1 hour at room temperature and washing all wells
twice with Wash Buffer, wells containing culture
sonicates were incubated with rabbit anti-luciferase
followed by HRP/anti-rabbit IgG conjugate for
detection of GST-luciferase fusion proteins. Wells
containing rGST standards were incubated with
HRP/Anti-GST Conjugate. All wells were developed
using TMB and the A450 of each well measured in
a 96-well plate reader.
Figure 3 is a photograph of the resulting detection
plate. Those wells which contain GST/luciferase are
evident by their strong positive signal. Wells exhibiting
rGST Control Anti-luciferase detection

for relative sensitivity of GST detection. 1 2 3 4 5 6 7 8 9 10 11 12

The TMB substrate absorbance readings for a wide

range of rGST Protein concentrations is shown in
Figure 2. As demonstrated in Figure 1, the Amersham
Biosciences GST 96-Well Detection Module
exhibits a strong signal for as little as 1 ng of rGST
per well. Products from Vendors X and Y failed to
yield signals appreciably greater than background
levels for that amount of rGST. In order to achieve
signals similar to that obtained for 1 ng detection
with the GST 96-Well Detection Module, the capture
plate from Vendor Y requires between 10 and 100 ng
of rGST in the sample and that from Vendor X
Figure 3. Screening of bacterial lysates for GST fusion protein expres-
requires over 100 ng of rGST. sion using the GST 96-Well Detection Module. rGST control (columns
1-3) = serial dilutions of rGST applied to the wells of a GST 96-Well
Screening bacterial lysates with fusion partner Capture Plate and detected by the standard protocol using HRP/Anti-
detection GST Conjugate described in the text. Anti-luciferase detection
(columns 4-12) = Lysates of randomly selected pGEX-6P-1/luciferase
To demonstrate that fusion proteins immobilized transformants detected with rabbit anti-luciferase and HRP/anti-rabbit
through their GST domains can be detected with IgG. All samples treated with TMB substrate for detection.

Life Science News 1, 1998 Amersham Biosciences

2
GST96detnr11.qxd (rev) 8/12/98 1:05 pm Page 3

I N N O V A T I O N S F O R U M

weak or no signal are thought to contain only GST

from parental pGEX-6P-1 vector expression.

Conclusions
The GST 96-Well Detection Module has been shown
to detect as little as 1 ng of recombinant GST with a
chromogenic substrate. That sensitivity is 10 to 100
times greater than capture plates from other vendors
which use immobilized glutathione. The added sensi-
tivity of the GST 96-Well Detection Module is
thought to be derived from the increased binding
capacity and affinity afforded by the polyclonal anti-
GST antibody coating the wells of the module’s cap-
ture plates. The resulting capture of GST fusion pro-
teins is very specific, and the pre-blocked plates
exhibit low, non-specific background binding even
from complex protein mixtures such as bacterial son-
icates. GST fusion proteins captured on the well sur-
face of the GST 96-Well Capture plates can be
detected using the HRP/Anti-GST Conjugate included
in the module. Antibodies directed against the fusion
partner also work effectively for detection of cap-
tured fusion proteins. Screening of as many as 96
samples per plate can be rapidly performed with the
GST 96-Well Detection Module.

References
1. Nilsson, J. et al., Protein Expression and Purification
11, 1-16 (1997).
2. Smith, D. B. and Johnson, K. S., Gene 67, 31-40 (1988).

ORDERING INFORMATION
GST 96-Well Detection Module five 96-well detections 27-4592-01
Related Products
MicroSpin™ GST Purification Module 50 purifications 27-4570-03

Life Science News 1, 1998 Amersham Biosciences