Anti-Penta-HIS (HIS) Biosensors - ForteBio

Document Title
Anti-Penta-HIS (HIS) Biosensors
Label-Free Quantitation of HIS-Tagged Proteins
Key Features
• Quantitate proteins containing a six-histidine
or penta-histidine peptide tag (HIS-tag)
• Designed for use in buffer, complex media,
column eluates, and cell lysates
• Binds HIS-tagged proteins even in the
presence of imidazole
• Easy to use, label-free, no wash protocol
Introduction
The six-histidine or penta-histidine peptide tag (HIS-tag) is a common
peptide tag fused to recombinant proteins during cloning.
Many tools have been developed enabling the tag to be used for
easy detection and purification of tagged proteins. The Anti-
Penta-HIS biosensor, for use on the Octet system, now provides a
rapid, label-free method for quantification of HIS-tagged proteins.
This biosensor comes with the highly specific Qiagen Penta-HIS
antibody pre-immobilized on the surface and is ready to use for
specific detection and quantitation of HIS-tagged proteins.
Quick facts
• Immobilization Chemistry: highly specific Qiagen Penta-HIS
antibody (Qiagen part no. 34660)
• Quantitation Dynamic Range: 0.25–200 µg/mL
• Compatible Matrix Types: buffers, complex media, column
eluates, and cell lysates
Intended Use
The biosensor is intended for the detection and quantitation of
HIS-tagged proteins. Proteins can be purified, in column eluates,
1A
0.80
1B
1.000
0.70
0.60
16 µg/mL
8 µg/mL
4 µg/mL
2 µg/mL
1 µg/mL
0.25 µg/mL
0.25 µg/mL
0 µg/mL
nm
0.50
0.40
Binding Rate
0.100
0.30
0.20
0.10
0.00
0 30 60 90 120 150 180 210 240 270 300
T ime (sec)
0.010
0.10 1.00
10.00
Concentration (µg/mL)
FIGURE 1A: Real-time binding data from a concentration series of HIS-tagged PAI
(Oxford Biomedical Research, part no. PI06) obtained using the Anti-Penta-HIS
biosensor on an Octet RED. The concentration series tested was 16, 8, 4, 2, 1, 0.5,
0.25 and 0 μg/mL.
FIGURE 1B: Calibration curve generated from binding data for HIS-tagged PAI using
the R Equilibrium algorithm.
1

Anti-Penta-HIS (HIS) Biosensors
Sample Type
Purified proteins
Samples containing imidazole
(elutes from NTA resin)
Serum-free cell culture
supernatants media
Serum-containing cell
culture supernatants
Bacterial cell pellet lysed by
freeze-thaw
Bacterial cell pellet lysed by
detergent
Minimum Recommended Dilution
In ForteBio Sample Diluent
Dilute into assay range as necessary
Dilute into assay range as necessary
1:10 dilution
1:100 dilution
1:10 dilution
Table 1: Recommended minimum dilution for each sample type. NOTE: In all cases
the matrix for the diluted samples, the calibrator and the biosensor hydration solution
should be matched as closely as possible.
Calculated Conc. µg/mL
1:100 dilution 0
80
60
40
20
CHO Cell Spent Media
ForteBio Sample Diluent
0 10 20 30 40 50 60
Expected Conc. µg/mL
FIGURE 2: Comparison of calculated concentrations for the quantitation of the
same HIS-tagged protein in ForteBio Sample Diluent and CHO cell spent media.
cell culture supernatants or cell lysates. Performance in each
matrix type should be optimized through appropriate sample
dilution and careful construction of a suitable calibration curve.
This biosensor is intended for research and manufacturing use
only and is not intended for diagnostic use in humans or animals.
Applications
The Qiagen Penta-HIS antibody (Qiagen part no. 34660) is preimmobilized
onto the biosensors and will bind specifically to a
HIS-tag attached to recombinant proteins (refer to the Qiagen
QIAexpress Handbook for more specific epitope information). This
binding is monitored in real time using the Octet system and can
be compared to the binding of a known calibrator to determine
concentration (Figure 1). Since the antibody-coated biosensors
will bind HIS-tagged protein in the presence of complex matrix
components or imidazole, the Anti-Penta-HIS biosensor can be
used to quantitate HIS-tagged proteins from Ni/NTA column
elutes, cell culture supernatants (Figure 2) and cell lysates after
the appropriate dilution into sample diluent (Table 1).
Typical Assay Parameters
• Sample Volume: 200 µL/well (post-dilution)
• Hydration Sample Volume: 200 µL/well (post-dilution)
• Flow rate: 1000 rpm
• Biosensor Hydration and Sample Plate Equilibration: 10 minutes
• Assay Time per Column of Samples: 3–5 minutes
Ordering information
Part No. UOM Description
18-5077 Tray One tray of 96 Anti-Penta-HIS (HIS)
biosensors
18-5078 Pack Five trays of 96 Anti-Penta-HIS (HIS) biosensors
18-5079 Case Twenty trays of 96 Anti-Penta-HIS (HIS)
biosensors
Note: additional materials are required to run these assays.
Please consult Technical Note 19 for full details.
Dip and Read Anti-Penta-HIS biosensors are compatible with all
Octet instruments including the new Octet 384 series. The latest
version of software includes a predefined protocol to make this
assay quick and easy to run and is available with 21 CFR Part 11
compliance tools.
For more information about ForteBio’s Octet platform for
label-free, real-time detection of biomolecular interactions,
applications, and services, visit www.fortebio.com or contact
us directly.
ForteBio, Inc.
1360 Willow Road, Suite 201
Menlo Park, CA 94025
t: 888.OCTET-QK
or 650.322.1360
ForteBio, UK, Ltd.
83 Victoria Street, Suite 407
London, SW1H 0HW, UK
t: +44-(0)20-31784425
www.fortebio.com
ForteBio (Shanghai) Co., Ltd.
917 Halley Road, Bldg 4
Zhangjiang High Tech Park
Shanghai, China 201203
t: +86-21-51320387
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