ProteoSpin™
Inclusion Body Isolation Kit
The
ProteoSpin™ Inclusion Body Isolation Micro Kit facilitates the
screening of E. coli clones that express recombinant proteins in
inclusion bodies. The kit includes reagents specially formulated to
achieve rapid and high-quality purification of inclusion bodies
using three processes:
-
Lysis of
bacterial cells to release inclusion bodies in solid form
-
Solubilization
of purified inclusion bodies
-
Purification of
the recombinant protein using SiC spin column chromatography
Downstream experiments include SDS-PAGE analysis, refolding
experiments, mass spectrometry, further purification and scale up.
The kit contains solutions to purify inclusion bodies from both
acidic and basic proteins using 1.5 mL bacterial culture harvested
at the end of the protein production period.
The
ProteoSpin™ Inclusion Body Isolation Micro Kit employs an innovative
separation material, silicon carbide (SiC). This proprietary
processed material acts as an ion exchanger. Each Inclusion Body
Isolation Micro Kit spin column contains 25 mg SiC with a protein
capacity of 2-50 mg and may vary depending on protein sample.
ProteoSpin Inclusion Body Isolation Kit Benefits
-
Three kits in
one for acidic and basic proteins
- The kit provides essential reagents for cell disruption,
inclusion body solubilization and purification using spin column
chromatography. The kit includes solutions and protocols for use
with either acidic or basic proteins.
-
Gentle
disruption procedure provides high purity final product
- Cell lysis is accomplished through non-ionic detergent chemical
disruption. Passing cells through a needle eliminates the need for
cell sonication or other forms of mechanical disruption, producing
protein often >95% pure. Proteins are then ready for SDS-PAGE, 2D
gels, mass spectrometry analysis or other procedures.
-
Convenient
process volume
- Analyze 1.5 mL of microbial cell culture yielding 2–50 mg of
protein.
-
Long shelf life
- The kit uses an enzyme-free process with stable solutions for
longer shelf life.
Fast
process time
- Process up to 12 samples in only 60 minutes.
Each
ProteoSpin™ Inclusion Body Isolation Micro Kit spin tube contains 25
mg SiC. Capacity per spin tube is approximately 2-50µg and may vary
depending on protein sample. The protocol was designed for use with
1.5 mL of bacterial cultures harvested at the end of the protein
induction period.
ProteoSpin™
Inclusion Body Isolation micro Kit Contents
Each
ProteoSpin™ Inclusion Body Isolation Micro Kit provides essential
components for use with the provided protocols.
-
Cell Lysis
reagent
-
Inclusion Body
solubilization reagent
-
Column
activation and wash buffer for acidic proteins
-
pH binding
buffer for acidic proteins
-
Column
activation and wash buffer for basic proteins
-
pH binding
buffer for basic proteins
-
Elution buffer
-
Neutralizer
-
ProteoSpin™ SiC
micro spin columns
-
Collection Tubes
(2 mL capacity)
-
Final Collection
Tubes (1.7 mL capacity)
-
Syringes (1 mL
capacity)
-
Needles (20
gauge)
-
ProteoSpin™
Inclusion Body Isolation Micro Kit application manual
-
Protocol
reference card for both acidic and basic procedures
Customer-supplied Reagents and Equipment
-
Benchtop
microcentrifuge
-
Micropipetters
Sterile, deionized water or Milli-Q water
Technical Note Abstract
A
simple time course experiment was carried out to determine the
minimum incubation time after induction needed to attain maximum
expression levels using the ProteoSpin™ Inclusion Body Isolation
Kit.
A
stock culture of an E. coli
expressor for a 40 kDa protein was streaked on a selection of agar
plates containing antibiotics and incubated. One colony was picked
and inoculated in a test tube with Luria and antibiotics. After
incubation, the solution was centrifuged and the pellet resuspended
in fresh solution and incubated until an OD600 of 0.6 was reached.
IPTG was added to induce protein expression. Aliquots were removed
at 3 hours following induction and then every 20 minutes.
The solution was centrifuged, cell lysis reagent was added to the
cell pellets, and inclusion body isolation was performed. Once the
final inclusion body pellets were obtained, solubilization reagent
was added to each tube. The inclusion body solution was run through
pre-activated ProteoSpin™ columns using the acidic protocol. The
flowthrough was discarded and the proteins were eluted twice using
elution buffer.
The purified protein samples were run on a 12.5% polyacrylamide gel.
The full-length recombinant protein started to appear between 40 and
60 minutes post induction, and continued to increase until a maximum
level was reached after 160 minutes. Thus, a minimum incubation time
of 3 hours after induction will produce a substantial amount of
protein.
Figure 1:
Time course study for expression of a 40 kDa protein. Expression was
induced with 0.4 mM IPTG and incubated at 37 °C for three hours.
Aliquots were removed at indicated intervals and purified. Proteins
made visible by Coomassie Blue staining.
Download the complete application notes:
Downloadable Application
Notes
Screening For Bacterial Clones Expressing Inclusion Body Proteins
A Time Course Induction Study of Recombinant Protein Expression in
E. coli
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