Purification of Tagged Proteins
Fast affinity purification of GSTand His-tagged proteins
New high-throughput filter plates ideal for small volumes
The expression and purification of recombinant proteins is
central for studying protein regulation, structure, function
and interactions. To accelerate and aid protein purification
and detection, the majority of recombinant proteins are
expressed as fusions with a peptide or protein tag. The
polyhistidine (His) tag and glutathione S-transferase (GST)
tag are the most popular and commonly used fusion tags.
We offer several affinity chromatography products for the
purification of His- and GST-tagged recombinant proteins
from bacterial, mammalian or baculovirus-infected insect
cells. These diverse products provide a range of options
to best suit your research needs.
The Thermo Scientific HisPur Ni-NTA, HisPur Cobalt and Pierce
Glutathione Products are available in the following formats:
•Bulk Resin: 10mL, 100mL, 500mL bottles
•Spin Columns: 0.2mL microcentrifuge, 1mL, 3mL columns
•Purification Kits: containing columns, buffers and collection tubes
•Chromatography Cartridges: compatible with automated liquidchromatography systems and syringe processing
•Large-volume and custom orders
•High-throughput 96-well filter plates (Figure 1)
Highlights:
•Compatible – use with Thermo Scientific Cell Lysis Reagents and
a variety of buffer additives
•Cost-effective – resin is economically priced and can be reused
at least five times
•Flexible – available in multiple formats including bulk resin, spin
columns and chromatography cartridges
•Easy to use – pre-formulated buffers available for kit formats
These products are easy to use and provide high capacity, yield
and purity at an exceptional price. The HisPur™ Ni-NTA or HisPur
Cobalt Resins contain metal-charged NTA-chelate immobilized
onto 6% agarose and provide excellent binding capacity and
performance for recombinant His-tagged protein purification. Use
cobalt for ultimate purity and Ni-NTA for stronger protein binding
and higher resin capacity (Figures 2 and 3).
18
www.thermoscientific.com/pierce
Figure 1. Ready-to-use 96-well filter plates for high-throughput small-volume
separation of tagged proteins. Each well in the plate has a binding capacity of
approximately 1mg of tagged protein.
Co2+
1
2
Ni2+
3
4
5
6
L
His-tagged β-gal
-135kDa
Figure 2. Use Thermo Scientific HisPur Cobalt Resin for high purity. HisPur
Cobalt Resin (Lane 1) was compared to other IMAC resins (Lanes 2–6). Cell
lysates containing over-expressed recombinant 6xHis-tagged protein were
prepared in Thermo Scientific B-PER Bacterial Protein Extraction Reagent
(Product # 78243) and protease inhibitors (Product # 78410). Protein concentrations
were determined by the Thermo Scientific Coomassie Plus Protein Assay
(Product # 23238). E. coli lysates containing over-expressed His-tagged
β-galactosidase were applied to 0.2mL bed volumes of each IMAC resin in a
spin-column format. Binding, wash and elution buffers were prepared and used
per each manufacturers’ instructions. The first elution fraction for each IMAC
resin was analyzed by SDS-PAGE and stained with Thermo Scientific Imperial
Protein Stain (Product # 24615). Protein purity was determined by densitometry:
Gel lanes were normalized to equivalent volume. Lane 1: HisPur Cobalt Resin,
Lane 2: Clontech cobalt resin, Lane 3: Sigma cobalt resin, Lane 4: GE Healthcare
Ni resin, Lane 5: Qiagen Ni-NTA resin, Lane 6: Ni-IDA and L= lysate load.
Vol. 15, Issue 1
Thermo Scientific
HisPur Ni-NTA
M
The newest offerings to our His- and GST-tagged protein purification
products are ready-to-use 96-well filter plates for high-throughput
small-volume separation of tagged proteins. This format is ideal for
applications such as expression library screening and optimization
of expression and purification conditions. Our affinity-tag purification
filter plates provide consistent well-to-well and plate-to-plate
reproducibility and are compatible with centrifugation and
vacuum-manifold systems for manual or automated purification.
Each well in the plate has a binding capacity of approximately
1mg of tagged protein.
Other Suppliers
L
Q
C
Ni-IDA
Ordering Information
Figure 3. Thermo Scientific HisPur Ni-NTA resin performs as well or better
than other suppliers’ nickel resins. Bacterial lysate (12mg total protein)
containing over-expressed 6xHis-green fluorescent protein (GFP) was applied
to HisPur Ni-NTA Resin (0.2mL) and purified by the batch method. The same
amount of total protein was applied to the Ni-NTA resin from Qiagen (Q) and
Clontech (C). An Ni-IDA resin was also examined. All samples were prepared
using the manufacturers’ instructions. Samples were analyzed by SDS-PAGE
and stained with Thermo Scientific GelCode Blue Stain Reagent (Product #
24590). Gel lanes were normalized to equivalent volume. M= molecular-weight
marker and L= lysate load.
Product #
88230
Description
HisPur Ni-NTA Spin Plates
2 plates
U.S.
Price
$310
16111
Pierce Glutathione Spin Plates
2 plates
$325
90095
HisPur Cobalt Spin Plates
2 plates
$321
Contains two 96-well filter plates (50µL resin bed/
well; 200µL of 25% slurry) and six wash/collection
plates.
Contains two 96-well filter plates (50µL resin bed/
well; 100µL of 50% slurry) and six wash/collection
plates.
Contains two 96-well filter plates (100µL/well; 400µL
of 25% slurry) and six wash/collection plates.
Pkg. Size
Please see our website for a complete listing of available formats and kits.
The Pierce® Glutathione Agarose contains glutathione immobilized
to 6% beaded agarose via a 12-atom spacer arm to the central
sulfhydryl group. Using a buffer containing reduced glutathione,
bound GST-fusion proteins are specifically eluted at high yield
(Figure 4).
Thermo
GE
Scientific Healthcare
kDa
M
L
FT
E
FT
E
Qiagen
FT
E
Clontech
FT
E
Sigma
FT
E
250
150
100
75
50
27
Protein Purification Technical Handbook
25
20
15
10
Figure 4. Thermo Scientific Pierce Glutathione Agarose delivers high
yield GST-fusion proteins. E. coli lysate (14.4mg total protein) containing
overexpressed GST was incubated with 50µL of glutathione resin from various
suppliers and purified per manufacturers’ instructions. Purity was assessed
by densitometry of the stained gel lanes. Elution fractions from all resins
analyzed resulted in > 90% purity for the target protein. M= Molecular
weight marker; L=Lysate load; FT=Flow-through; E=Elution.
This handbook provides protocols and
technical and product information to help
maximize results for protein purification.
It also includes background and troubleshooting advice for covalent coupling of
affinity ligands to chromatography supports,
avidin:biotin-binding, affinity purification of
antibodies, IP and co-IP, affinity procedures
for contaminant removal, and related
procedures. 1602015
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.
19