Q4_Test_Epic Cell based SS.qxd - Corning Incorporated

Achieving Similar Quality Results with
Lower Amounts of Protein in Corning ® Epic ®
Biochemical Assays
SnAPPShots
A brief technical report
from the Corning
Development Group
Thomas Bunch and Alice Gao
Corning Incorporated
Life Sciences
Kennebunk, Maine
Introduction
For many laboratories, procuring, manufacturing, or purifying
large quantities of a purified protein to perform a highthroughput
label-free biochemical assay can be problematic.
As a result, the following work was performed to demonstrate
the validity and versatility of using lower amounts of
protein when performing a biochemical screen using the
Corning Epic Technology, a label-free detection system that
uses ANSI/SBS standard 384 well microplates. The Epic
technology enables the detection of molecules binding directly
to an immobilized target without the use of fluorescent
or radioactive tags. Typically in a label free assay, protein or
peptides are immobilized on the surface of each well in a
microplate. This is a critical step because the immobilization
level of proteins or peptides often determines the signal level
of binding events, especially for molecules below 300 Daltons.
The higher the immobilization level of protein, the higher
the corresponding signal of binding events. To achieve a high
immobilization level, the amount of protein required can
sometimes become an issue. For example, immobilization
levels of 2,000 picometers (pm) or above are considered
reasonable for detecting small molecule binding. This
immobilization level is usually achieved by using a concentration
of 50 µg/mL protein in 15 µL of buffer. This
requires the use of 750 ng of protein per well with a total
of 288 µg per plate. Depending on the protein, this amount
can be difficult or expensive to obtain.
In this study, a strategy was developed to reduce the amount
of protein required while maintaining an immobilization
level that provides robust results in the Epic biochemical
assay described. Proteins with a wide range of pI values were
included in this study to demonstrate the versatility of this
approach.
Materials and Methods
Reagents
All proteins, including trypsin (Cat. No. T0303), streptavidin
(Cat. No. S4762), carbonic anhydrase (Cat. No. C2522),
lysozyme (Cat. No. L6876) and papain (Cat. No. P4762)
were obtained from Sigma-Aldrich. Each of these proteins
were made into stock solutions containing 5 mg/mL, dispensed
into aliquots and kept frozen at -20°C prior to any
experiments in the following buffers. For trypsin, streptavidin
and carbonic anhydrase, optimal immobilization was
achieved with 20 mM sodium acetate buffer (Thermo-
Fisher, Cat. No. BP333-500) at pH 5.5, 5.5 and pH 5.1,
respectively. For lysozyme and papain, optimal immobilization
was achieved with 20 mM sodium phosphate at pH 8.0
(Sigma, Cat. No. 82593). Phosphate buffered saline (PBS),
pH 7.4, was used for washing the plates and was purchased
from Sigma (Cat. No. P3813).
Assay Procedure:
All immobilizations were performed overnight at 4°C in
Epic biochemical microplates (Corning, Cat. No. 5041)
covered with foil. Protein was added to the wells of each
microplate using a Matrix Impact 2 16-channel automated
pipettor (Cat. No. 2061). After overnight immobilization,
plates were washed using a Biotek ELx405 TM plate washer
with PBS, pH 7.4. After washing, 15 µL of PBS/0.1%
DMSO was added to each well, and the immobilization
levels were measured using the Epic ® reader.
Data Analysis
The graphs were plotted using GraphPad Prism software.

Results
The first approach for minimizing the amount of immobilized
protein required to perform the assay was to reduce
the volume of the protein solution added to each well. The
immobilization levels of three frequently used proteins (carbonic
anhydrase, streptavidin and trypsin) using different
protein sample volumes per well were compared, while
keeping the protein sample concentrations consistent at 50
µg/mL. The optimal immobilization pH values of 5.5, 5.5
and 5.1 were used for carbonic anhydrase (CA), streptavidin
(SA) and trypsin, respectively. As summarized in Figure 1A,
there is a corresponding drop in immobilization levels of
approximately 20% for all three proteins when the volume
was reduced from 15 µL to 5 µL at the standard protein
concentration of 50 µg/mL. Therefore, a simple reduction
in protein sample volume is not a viable solution to reduce
protein consumption.
In the following test the protein concentration was increased
from 50 µg/mL to 75 µg/mL and the volume decreased for
addition to the well was evaluated an alternative method for
reducing the protein required to perform the assay. As shown
in Figure 1B, the slight increase in protein concentration to
75 µg/mL resulted in more consistent immobilization levels
across all volumes tested when compared to the results
achieved using 50 µg/mL protein concentration. A small
immobilization decrease of ~10% was observed using 5
µL/well as compared to 15 µL/well. A similar trend was
observed for all 3 proteins tested. A one sample t-test was
performed for each protein, comparing the immobilization
signals obtained from the standard condition with 15 µL of
50 µg/mL protein (equal to 750 ng per well protein consumption)
and those from the new condition with 5 µL of
75 µg/mL (equal to 375 ng per well protein consumption).
The results showed that there is no significant difference
between the immobilization levels obtained under these two
conditions (t [24] 0.05). These results suggest that a
strategy of reducing volume while increasing protein concentration
slightly is a viable option for controlling protein
consumption.
The three proteins tested above all require low pH for
immobilization. To better understand whether the approach
to optimize protein binding would apply to all proteins, two
proteins that typically require high pH for immobilization,
papain and lysozyme, were evaluated in a similar approach.
The optimal immobilization for these two proteins typically
is observed at pH 8.0. Therefore, sodium phosphate buffer
was used to dilute the proteins to concentrations of 75 µg/mL
and 50 µg/mL. Volumes of 15, 10 and 5 µL/well were evalu-
A. B.
Figure 1. Comparison of protein immobilization levels (pm) of carbonic anhydrase (CA), streptavidin (SA) and trypsin after the addition of either 15, 10 or
5 µL protein solution at either (A) 50 µg/mL protein concentration or (B) 75 µg/mL protein concentration. The standard assay protocol recommends
15 µL of 50 µg/mL protein solution to be added to each well during the immobilization ste, which is equivalent to 750 ng total protein used per well.
Results are representative of 3 independent experiments (N = 24).

A. B.
Figure 2. Comparison of protein immobilization levels (pm) of papain or lysozyme after the addition of either 15, 10 or 5 µL protein solution at either
(A) 50 µg/mL protein concentration or (B) 75 µg/mL protein concentration. Results are representative of 3 independent experiments (N = 24).
ated as described and the results are summarized in Figure 2.
For papain, the observed trend was similar to the proteins
that require acidic pH for immobilization. The immobilization
level at the reduced volume of 5 µL was equivalent to
that at the standard volume of 15 µL when the protein
concentration was increased from 50 µg/mL to 75 µg/mL.
Lysozyme, on the other hand, did not follow this trend. A
significant reduction in the immobilization level was observed
when the volume was reduced from 15 µL to 5 µL regardless
of the lysozyme protein concentration. Reducing the
volume from 15 to 10 µL for this protein gave a similar
signal (data not shown), suggesting that it is possible to
still achieve quality results while conserving protein.
Conclusions
This study demonstrates that similar Epic ® label-free assay
results can be achieved by using 50% less protein during
the immobilization step compared to the standard protocol.
The 50% reduction in protein consumption was achieved
by lowering the volume used during immobilization from
15 µL to 5 µL while simultaneously increasing the concentration
of the protein immobilized from 50 µg/mL to
75 µg/mL. This approach works well for 3 proteins that require
a low or high pH for immobilization and for 1 of the 2
proteins that require high pH conditions for immobiliation,
leading to reduced protein consumption needed to run an
Epic biochemical assay.

For additional product or technical information, please visit www.corning.com/lifesciences
or call 1.800.492.1110. Outside the United States, please call 978.442.2200.
Corning Incorporated
Life Sciences
Tower 2, 4th Floor
900 Chelmsford St.
Lowell, MA 01851
t 800.492.1110
t 978.442.2200
f 978.442.2476
www.corning.com/lifesciences
Worldwide
Support Offices
A S I A / P A C I F I C
Australia/New Zealand
t 0402-794-347
China
t 86 21 2215 2888
f 86 21 6215 2988
India
t 91 124 4604000
f 91 124 4604099
Corning and Epic are registered trademarks of Corning Incorporated, Corning, NY.
All other trademarks in this document are the property of their respective owners.
Corning Incorporated, One Riverfront Plaza, Corning, NY 14831-0001
Japan
t 81 3-3586 1996
f 81 3-3586 1291
Korea
t 82 2-796-9500
f 82 2-796-9300
Singapore
t 65 6733-6511
f 65 6861-2913
Taiwan
t 886 2-2716-0338
f 886 2-2516-7500
E U R O P E
France
t 0800 916 882
f 0800 918 636
Germany
t 0800 101 1153
f 0800 101 2427
The Netherlands
t 31 20 655 79 28
f 31 20 659 76 73
United Kingdom
t 0800 376 8660
f 0800 279 1117
All Other European
Countries
t 31 (0) 20 659 60 51
f 31 (0) 20 659 76 73
L AT I N A M E R I C A
Brasil
t (55-11) 3089-7419
f (55-11) 3167-0700
Mexico
t (52-81) 8158-8400
f (52-81) 8313-8589
© 2011 Corning Incorporated Printed in U.S.A. 8/11 POD CLS-AN-166