NBC/CBED Manual MAXI F
Application Manual
CBED (Concentration, Buffer Exchange
and Desalting) MAXI KIT
DETERGENT CLEAN-UP MAXI KIT
For Use with P/Ns 17000 and 17100
Table of Contents
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
GENERAL INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
ION EXCHANGE CHROMATOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
SILICON CARBIDE AS AN ION EXCHANGER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
ISOELECTRIC POINT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
REMOVAL OF DETERGENTS FROM BIOLOGICAL SAMPLES . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
PROTEOSPIN™ CBED MAXI KIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
CBED Maxi Kit Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
ProteoSpin™ CBED Maxi Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Recommended Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Customer-Supplied Reagents and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Overview of CBED Maxi Kit Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Choosing a ProteoSpin™ Procedure: Acidic or Basic Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
CBED Protocol for Acidic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
CBED Protocol for Basic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
PROTEOSPIN™ DETERGENT CLEAN-UP MAXI KIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
Detergent Clean-up Maxi Kit Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
ProteoSpin™ Detergent Clean-up Maxi Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Recommended Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Customer-Supplied Reagents and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Detergent Clean-Up Protocol for Acidic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Detergent Clean-Up Protocol for Basic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Appendix 1. Optional Elution Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Appendix 2. Proteins with Established Isoelectric Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43
Appendix 3. Frequently Asked Questions and Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
Appendix 4. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
2
BIOTEK
CORPORATION
General Introduction
Norgen’s ProteoSpin™ Maxi Kits include the CBED (Concentration, Buffer
Exchange and Desalting) Kit and the Detergent Clean-up Kit. The maxi columns
contained in these kits are specifically designed for rapid protein purification and
isolation using spin-column chromatography technology. The Maxi Kits can process
samples containing 0.25 mg to 8 mg of protein. The technology used in these kits is
based on Norgen’s proprietary chromatography resin, silicon carbide (SiC). The SiC
acts as an ion exchanger that is very efficient in concentrating protein solutions, in
lowering salts, and in removing greater than 95% of the detergents present.
1
The ProteoSpin™ CBED Maxi Kit provides a fast and simple procedure for concentrating large volumes of dilute protein solutions for buffer exchange, and for removing different types of salts from protein samples. The kit is highly efficient in
removing many different salts commonly used in the laboratory including, but not
limited to MgCl2, NaCl, KCl, CaCl2, LiCl, and CsCl. The simultaneous removal of
salts while concentrating a dilute protein solution makes the kit a convenient method
for preparing proteins before running many downstream applications such as SDSPAGE and iso-electric focusing. These large columns are frequently used to prepare
protein samples for structural analysis where larger amounts of protein are needed,
such as X-ray crystallography, NMR spectroscopy and other applications.
BIOTEK
CORPORATION
3
General Introduction
Concentrating and desalting protein solutions is a key step in protein sample preparation. Conventional salt removal techniques, including dialysis, spin dialysis, gel filtration, precipitation and solid phase extraction, are time-consuming and, in the case of
dialysis, may result in dilution of the protein sample. The use of the Proteospin™
CBED Maxi Kit results in high-quality protein preparations with the proteins concentrated, buffer exchanged, and free of the original sample salts.
The ProteoSpin™ Detergent Clean-Up Maxi Kit provides a fast and simple procedure
for removing SDS and other detergents from protein samples. Detergents are extensively used to prepare protein samples; however, detergents must be removed prior to
downstream analysis because of their undesirable effects. For example, detergents can
often cause poor results in instruments such as mass spectrometers due to large numbers of contaminating peaks and may actually cause physical harm to the instrument.
In addition, detergents interfere with purification procedures such as chromatography
and gel electrophoresis as well as with structural characterization techniques such as
crystallography and amino acid sequence analysis.
4
BIOTEK
CORPORATION
General Introduction
Ion Exchange Chromatography
In ion exchange chromatography, ions that are electrostatically bound to an insoluble
and chemically inert matrix are reversibly replaced by ions in solution. The matrix can
be either an anion exchanger, consisting of positively charged groups that reversibly
bind anions in solution, or it can be a cation exchanger bearing negatively charged
groups that bind to mobile cations. Proteins are complex molecules that can have an
overall positive or negative charge that depends on their amino acid composition and
the pH of their solutions. In this case, any given protein will bind to anion or cation
exchangers depending on the net charge of the protein. When purifying a protein, the
pH and salt concentration of the buffer solution (in which the protein is dissolved) are
chosen to immobilize the desired protein on the selected ion exchanger. The impure
protein solution is applied to a column that contains the ion exchanger, and has been
washed and equilibrated with the buffer solution. The proteins bind to the matrix and
are eluted by either changing the salt concentration or the pH.
BIOTEK
CORPORATION
5
General Introduction
Silicon Carbide as an Ion Exchanger
The chromatographic resin used in the ProteoSpin™ Maxi Kits consists of silicon carbide (SiC), a man-made material noted for its hardness (second only to diamond) and
high resistance to chemical change. These properties make SiC highly suitable for spin
column chromatography even at extremes of pH. When processed appropriately, SiC
becomes an effective cation exchanger for the purpose of purifying macromolecules.
The surface of SiC is negatively charged and can bind positively charged macromolecules. SiC has poor affinity to monovalent and divalent cations, also making it an
effective resin for the removal of salts.
Soluble proteins bind to SiC through the interaction between the positively charged
side-groups on the polypeptide chain and the negative charges on the SiC surface. As a
cation exchanger, SiC has an estimated effective pKa ≈ 2 making the resin negatively
charged over a wide range of pH. Therefore, with the exception of those peptides that
are highly acidic, most soluble polypeptides can be bound to SiC.
6
BIOTEK
CORPORATION
General Introduction
Isoelectric Point
Proteins generally contain a number of ionizable groups with a variety of pKa values.
For each protein in a solution, there is a particular pH where the negative charges on
the molecule exactly balance the positive charges, therefore giving the protein zero net
charge. This is known as the isoelectric point, or pI of the protein. The pI of a protein
determines the ideal conditions for its purification by ion exchange chromatography.
For example, based on pI, one can determine whether the pH of a binding buffer and
an elution buffer are suitable for a particular protein. Isoelectric points for a number of
proteins have already been determined. If the pI of a protein of interest is unknown, a
theoretical pI can be determined using web-based tools found at the Expasy site
http://us.expasy.org/tools/pi_tool.html. Another useful pI-predictor method can be
found at http://www.up.univ-mrs.fr/~wabim/d_abim/compo-p.html. It should be noted,
however, that the apparent pI of a protein as revealed in isoelectric focusing studies
may differ significantly from its theoretical pI.
BIOTEK
CORPORATION
7
General Introduction
Removal of Detergents from Biological Samples
Detergents are surface-active agents (surfactants) that contain both a hydrophobic portion and a hydrophilic portion. In aqueous solutions, detergents may form stable structures called micelles with their hydrophilic portions facing the aqueous environment
and their hydrophobic portions being hidden in the core.
The charge of the hydrophilic portion enables the classification of detergents into four
major groups; anionic, cationic, zwitterionic and non-ionic. Anionic and cationic detergents can be used for processes ranging from solubilizing membrane proteins under
non-denaturing conditions to the denaturing conditions of running SDS-PAGE electrophoresis. Common ionic detergents include SDS and sodium deoxycholate.
Zwitterionic detergents, such as CHAPS, protect the native state of the protein while
preventing protein aggregation during purification procedures. Zwitterionic detergents
have low denaturing features whereas non-ionic detergents are non-denaturing. This
makes non-ionic detergents suitable for solubilizing membrane proteins without altering biological activity by maintaining the secondary and tertiary structures of the protein. However, these non-ionic detergents, including NP-40, Triton X-100 and Tween
20, are less effective at disrupting protein aggregation. Conventional methods for
detergent removal include hydrophobic adsorption, gel chromatography, dialysis, ionexchange chromatography and precipitation techniques. These methods are time-consuming and tedious and may also result in the loss of protein sample.
8
BIOTEK
CORPORATION
General Introduction
The ProteoSpin™ Detergent Clean-Up Maxi Kit is an innovative and user-friendly
method that can remove greater than 95% of detergents (based on Triton X-100 and
SDS measurements) from protein samples while maintaining high protein recovery.
The kit is able to remove all types of detergents including ionic, non-ionic and zwitterionic. It is designed to remove detergents in protein solutions either in their free form
or bound form as when complexed with the protein. Detergent molecules that are free
in the solution cannot bind to SiC if the detergent is negatively charged because it is
repelled by the negative charges on the surface of SiC. Positively charged detergents,
however, are expected to preferentially interact with SiC, causing proteins to bind
poorly.
By virtue of the polar and nonpolar regions in a detergent molecule, detergents can
partition in both hydrophobic and hydrophilic environments. Most detergents unbind
from proteins in an aqueous environment in the presence of alcohols, freeing the proteins to interact with water molecules. If the solution is now allowed to move along a
chromatographic column containing the SiC resin, the protein molecules bind to the
resin while the detergent moves along with the liquid phase. The captured protein can
then be eluted into a different buffer free of detergents.
BIOTEK
CORPORATION
9
ProteoSpin™ CBED Maxi Kit
ProteoSpin™ CBED Maxi Kit Benefits
Basic Features
Benefits
Fast and easy processing
Efficient processing of multiple samples in
about 30 minutes using an easy-to-use
protocol.
No mixing or formulation
Solutions are provided for both acidic and
basic proteins with no need for formulation.
No molecular weight cutoff
Based on ion exchange mechanism, a broad
size range of proteins (from peptides to large
proteins) can be processed.
Proteins bind while salts are
discarded in flowthrough
SiC has an inherent low affinity for salt and a
high affinity for proteins, hence it provides salt
removal and buffer exchange simultaneously.
Suitable for downstream
applications
Final elution performed with volatile or nonvolatile buffers suitable for:
• Mass spectrometry
• SDS-PAGE
• Isoelectric focusing
• X-ray crystallography
• NMR spectroscopy
Maxi columns are provided
Allows for binding capacities up to 8 mg.
10
BIOTEK
CORPORATION
2
ProteoSpin™ CBED Maxi Kit
ProteoSpin™ CBED Maxi Kit Components
•
•
•
•
•
•
•
Column Activation and Wash Buffer (Acidic)
Column Activation and Wash Buffer (Basic)
pH Binding Buffer (Acidic)
pH Binding Buffer (Basic)
Elution Buffer
Neutralizer
ProteoSpin™ SiC Maxi Spin Columns (filled with SiC) inserted
into 50mL collection tubes
• Final Elution Tubes (50 mL)
• ProteoSpin™ CBED Maxi Kit Application Manual
• ProteoSpin™ CBED Maxi Kit Protocol Card
Recommended Storage Conditions
For unopened solution containers, the reagents should remain stable for 12
months when stored at room temperature. Once opened, all solutions,
except for the two binding buffers, should be stored at 4°C when not in use.
The binding buffers should remain at room temperature with the lids tightly
closed. Salt crystal formation may occur when stored at 4°C. If crystals
are visible, bring the entire bottle to room temperature and mix gently to
redissolve.
BIOTEK
CORPORATION
11
ProteoSpin™ CBED Maxi Kit
Customer-Supplied Reagents and Equipment
You must have the following in order to use the ProteoSpin™ CBED Maxi Kit:
•
•
•
•
Micropipettors
pH Indicator paper
Benchtop centrifuge (capable of spinning the 50mL conical tubes)
Other elution buffers (optional)
Procedure
Overview of CBED Maxi Kit Procedures
The kit utilizes spin columns containing SiC, a cation exchanger that largely depends
on pH for its reversible binding capability and is affected minimally by high salt concentrations. Binding occurs if the protein or proteins of interest retain a net positive
charge at the binding pH. Non-specifically bound materials such as salts are washed
from the column and the bound protein is eluted into a small volume of elution buffer.
The process results in an effective concentration and desalting of the protein. Each spin
column is able to concentrate and desalt up to 8 mg of acidic or basic protein. Two
protocols are described, depending on whether the protein of interest is acidic or basic.
12
BIOTEK
CORPORATION
ProteoSpin™ CBED Maxi Kit
Choosing a ProteoSpin™ Procedure: Acidic or Basic Protocol
The ProteoSpin™ CBED Maxi Kit comes with solutions for concentrating and desalting both acidic and basic proteins. The user must select one or the other protocol that
is appropriate to the protein being purified. In theory, the protocol for acidic proteins
should apply to a majority of acidic and basic proteins since the resin is a cation
exchanger. That is, all proteins with pI greater than the binding pH at 4.5 should bind.
Basic proteins, however, bind strongly when they are used under these conditions,
making their elution quite inefficient. Therefore, for soluble basic proteins, a different
condition for binding the protein to the resin has been developed. For the purposes of
this kit, a protein whose pI is less than 8 will be treated as acidic and will use the
acidic protocol; otherwise, use the basic protocol. Figure 1 illustrates how to select
which procedure to follow for a particular protein given the pI of that protein.
Binding pH
Figure 1. Choosing a procedure based on the isoelectric point (pI)
BIOTEK
CORPORATION
13
CBED Protocol for Acidic Proteins
Protocols
The following procedures describe the concentration and desalting of acidic or basic
proteins in either buffered or unbuffered (dissolved in water) solutions.
Note: All centrifugation steps in these procedures are carried out at 1,000 x g in a
benchtop centrifuge. Please check your centrifuge’s user manual to determine the
appropriate speed that gives 1,000 x g. Performance of the kit is not affected by temperature, and thus the procedure may be performed at room temperature, 4ºC, or on
ice. The user must take discretionary measures, such as chilling samples in ice, to preserve biological activity.
CBED Protocol for Acidic Proteins
Proteins with isoelectric points (pI) of less than 7 are by definition acidic proteins.
However, for purposes of using the kit, the Protocol for Acidic Proteins applies to any
protein whose pI is less than 8.0. Proteins with pI higher than 8.0 are purified using
the Protocol for Basic Proteins. If the pI of the protein being purified is not known,
the theoretical pI may be calculated using the web-based applications at
http://us.expasy.org/tools/pi_tool.html or
http://www.up.univmrs.fr/~wabim/d_abim/compo-p.html.
14
BIOTEK
CORPORATION
CBED Protocol for Acidic Proteins
Protocols
A. Sample Preparation
Note: In preparing your sample for column binding by centrifugation, you will mix your
protein solution with the pH Binding Buffer (Acidic) provided in the kit to make proper
adjustment of the pH. Make sure that all particulates in your sample have been removed by
either filtration or centrifugation. The column reservoir has a capacity for 20 mL hence multiple centrifugations will be required for larger volumes.
1.
Obtain your protein sample and determine its volume.
2.
Depending on your starting protein solution, you will require a certain volume of the
pH Binding Buffer (Acidic) to adjust the pH of your protein solution to 4.5. If the
starting protein solution is in water, then add one part of the pH Binding Buffer
(Acidic) to 49 parts of your protein solution. However, if the starting protein solution
already contains a buffer, you may need greater volumes depending on your sample’s
buffer type and strength. Table 1 (page 16) serves only as guideline for the amount of
pH Binding Buffer (Acidic) to use for every milliliter volume of a protein solution in a
100 mM buffer to obtain pH 4.5. Please check the pH after mixing and add more if
necessary to obtain the desired pH.
Please note that if the protein solution is already at the desired pH or lower, do not
add any binding buffer.
BIOTEK
CORPORATION
15
CBED Protocol for Acidic Proteins
Table 1. pH Adjustment for Acidic Proteins
Starting pH of Solution
4
5
6
7
8
9
10
11
12
Volume of pH Binding Buffer (Acidic) per
milliliter of protein solution (based on
100mM buffered solution)
0
20 µL
20 µL
20 µL
50 µL
80 µL
80 µL
80 µL
100 µL
3. Set aside until the protein binding step.
16
BIOTEK
CORPORATION
CBED Protocol for Acidic Proteins
B. Column Activation
1. Obtain a spin column with its 50 mL conical collection tube. Unscrew the cap.
2. Apply 5 mL of Column Activation and Wash Buffer (Acidic) to the column. Screw
the cap back on LOOSELY.
3. Centrifuge at 1,000 x g for two minutes.
Note: Start timing spins only after centrifuge has reached desired speed.
4. Repeat steps 2 and 3 to complete the column activation step. Discard the flowthrough.
C. Protein Binding
During the binding step, the protein solution is passed through the resin bed aided
by centrifugation. The protein is captured as it comes in contact with the resin. For
most protein samples tested, the centrifugation time provides sufficient contact time
for the protein to bind.
1. Apply the protein sample (from the Sample Preparation step) onto the column and
centrifuge for two minutes. A maximum of 20 mL can be added at one time.
2. Discard the flowthrough. Reassemble the spin column with its collection tube.
Note: You can save the flowthrough in a fresh tube for assessing your protein's
binding efficiency.
BIOTEK
CORPORATION
17
CBED Protocol for Acidic Proteins
3. Depending on your sample volume, repeat steps 1 and 2 until the entire protein
sample has been applied to the column.
4. Discard any remaining flowthrough and reassemble the spin column with its collection tube.
D. Column Wash
This step removes non-specifically bound debris from the column.
1. Apply 10 mL of Column Activation and Wash Buffer (Acidic) to the column and
centrifuge for two minutes.
2. Discard the flowthrough and reassemble the spin column with its collection tube.
3. Add another 10 mL of Column Activation and Wash Buffer (Acidic) to the column
and centrifuge for two minutes.
4. Inspect the column and ensure that the liquid has passed through into the collection
tube. There should be no liquid in the column. If necessary, spin an additional two
minutes to dry.
18
BIOTEK
CORPORATION
CBED Protocol for Acidic Proteins
E. Protein Elution
The Elution Buffer that is supplied is 50 mM sodium phosphate pH 12.5. Please refer to
Appendix 1 (Optional Elution Buffers) for a list of alternate elution solutions that have been
tested with the kit. The proteins in the buffer should be neutralized immediately. It is
therefore recommended that the Neutralizer that is provided is pre-added to the elution tube
(fresh 50 mL conical tube). However, this is not needed for the first elution. For the second
elution, 0.3 mL of neutralizer is needed. Note: Please verify the pH of the first elution and
adjust with Neutralizer if required.
1. Add 300 µL Neutralizer, if desired (not required for 1st elution), to a fresh
50 mL collection tube (supplied).
2. Transfer the spin column, with bound protein, into the 50 mL collection tube from
step 1.
3. Apply 4 mL of Elution Buffer to column and centrifuge to elute bound protein.
4. A second elution is optional (another 4 mL). Repeat Steps 1 to 3 if desired.
Keep separate from the 1st elution.
Note: Approximately 90% of bound protein is recovered in the first two elutions. If desired,
a third elution may be carried out. This should be collected into a different tube (to which
Neutralizer is pre-added) to prevent dilution of the first two elutions.
Protein samples are now ready for downstream applications.
BIOTEK
CORPORATION
19
CBED Protocol for Basic Proteins
CBED Protocol for Basic Proteins
Proteins with isoelectric points (pI) of less than 7 are by definition acidic proteins. However,
for purposes of using the kit, the Protocol for Acidic Proteins applies to any protein whose pI
is less than 8.0. Proteins with pI higher than 8.0 are purified using the Protocol for Basic
Proteins. If the pI of the protein being purified is not known, the theoretical pI may be calculated using the web-based applications at http://us.expasy.org/tools/pi_tool.html or
http://www.up.univmrs.fr/~wabim/d_abim/compo-p.html.
Note: All centrifugation steps in these procedures are carried out at 1,000 x g in a benchtop
centrifuge. Please check your centrifuge’s user manual to determine the appropriate speed
that gives 1,000 x g. Performance of the kit is not affected by temperature, and thus the procedure may be performed at room temperature, 4ºC, or on ice. The user must take discretionary measures, such as chilling samples in ice, to preserve biological activity.
A. Sample Preparation
Note: In preparing your sample for column binding by centrifugation, you will mix
your protein solution with the pH Binding Buffer (Basic) provided in the kit to make
proper adjustment of the pH. Make sure that all particulates in your sample have been
removed by either filtration or centrifugation. The column reservoir has a capacity for
20 mL hence multiple centrifugations will be required for larger volumes.
20
BIOTEK
CORPORATION
CBED Protocol for Basic Proteins
1. Obtain your protein sample and determine the volume.
2. Depending on your starting protein solution, you will require a certain volume of
pH Binding Buffer (Basic) to adjust the pH of your protein solution to 7.0. If the
starting protein solution is in water, then add one part of the pH Binding Buffer
(Basic) to 49 parts of the protein solution. However, if the starting protein solution
is in a buffer, you may need greater volumes depending on the buffer type and
strength. Table 2 below serves only as guideline for the amount of pH Binding
Buffer (Basic) to use for every milliliter volume of a protein solution in a 100 mM
buffer to obtain pH 7. Please check the pH after mixing and add more if necessary
to obtain the desired pH.
Table 2. pH Adjustment for Basic Proteins
Starting pH of Solution
Volume of pH Binding Buffer (Basic) per mL of protein solution (based on 100 mM buffered solution)
4
5
6
7
8
9
10
11
12
150 µL
80 µL
80 µL
0 µL
60 µL
60 µL
60 µL
80 µL
80 µL
3. Set aside until the protein binding step.
BIOTEK
CORPORATION
21
CBED Protocol for Basic Proteins
B. Column Activation
1. Obtain a spin column with its 50 mL conical collection tube. Unscrew the cap.
2. Apply 5 mL of Column Activation and Wash Buffer (Basic) to the column. Screw the
cap back on LOOSELY.
3. Centrifuge for two minutes at 1,000 x g.
Note: Start timing only after centrifuge has reached desired speed
4. Repeat steps 2 and 3 to complete the column activation step. Discard the flowthrough.
C. Protein Binding
During the binding step, the protein solution is passed through the resin bed aided by centrifugation. The protein is captured as it comes in contact with the resin. For most protein samples
tested, the centrifugation time provides sufficient contact time for the protein to bind.
1. Apply the protein sample (from the Sample Preparation step) onto the column and
centrifuge for two minutes. A maximum of 20 mL can be added at one time.
2. Discard the flowthrough. Reassemble the spin column with its collection tube.
Note: You can save the flowthrough in a fresh tube for assessing your protein's
binding efficiency.
22
BIOTEK
CORPORATION
CBED Protocol for Basic Proteins
3. Depending on your sample volume, repeat steps 1 and 2 until the entire protein
sample has been applied to the column.
4. Discard any remaining flowthrough and reassemble the spin column with its collection tube.
D. Column Wash
This step removes non-specifically bound debris from the column.
1. Apply 10 mL of Column Activation and Wash Buffer (Basic) to the column and
centrifuge for two minutes.
2. Discard the flowthrough and reassemble the spin column with its collection tube.
3. Add another 10 mL of Column Activation and Wash Buffer (Basic) to the column
and centrifuge for two minutes.
4. Inspect the column and ensure that the liquid has passed through into the collection
tube. There should be no liquid in the column. If necessary, spin an additional two
minutes to dry.
BIOTEK
CORPORATION
23
CBED Protocol for Basic Proteins
E. Protein Elution
The Elution Buffer that is supplied is 50 mM sodium phosphate pH 12.5. Please refer to
Appendix 1 (Optional Elution Buffers) for a list of alternate elution solutions that have been
tested with the kit. The proteins in the buffer should be neutralized immediately. It is therefore recommended that the Neutralizer that is provided is pre-added to the elution tube (fresh
50 mL conical tube). For all elutions, 0.3 mL of Neutralizer is needed
1. Add 300 µL Neutralizer to a fresh 50 mL collection tube (supplied).
2. Transfer the spin column, with bound protein, into the 50 mL collection tube from
step 1.
3. Apply 4 mL of Elution Buffer to column and centrifuge to elute bound protein.
4. A second elution is optional (another 4 mL). Repeat Steps 1 to 3 if desired. Keep
separate from the 1st elution.
Note: Approximately 90% of bound protein is recovered in the first two elutions. If
desired, a third elution using Elution Buffer may be carried out. This should be collected into a different tube (to which Neutralizer is pre-added) to prevent dilution of
the first two elutions.
Protein samples are now ready for downstream applications.
24
BIOTEK
CORPORATION
ProteoSpin™ Detergent Clean-Up Maxi Kit
ProteoSpin™ Detergent Clean-Up Maxi Kit Benefits
Basic Features
Benefits
Fast and easy processing
In contrast to longer dialysis procedures, the
easy-to-use protocol allows processing of a
number of samples in about 40 minutes.
A complete kit for acidic and
basic protein samples
The kit contains essential solutions for processing detergent-containing samples from
0.25 - 8 mg of protein. Solutions are provided for processing both acidic and basic proteins.
No detergent carryover
Unlike HPLC procedures, each sample is
processed individually using its own spin
tube providing no sample carryover or
column bleed from sample to sample.
Usable with a variety of
detergents
This kit can be used to remove detergents
including, but not limited to, SDS, Triton X100, CHAPS, NP-40 and Tween 20.
Efficient detergent removal
Removes more than 95% of detergents for
many proteins, allowing tryptic digestion
without residual detergents that interfere with
trypsin digestion.
Maxi columns are provided
Allows for binding capacities up to 8 mg.
BIOTEK
3
CORPORATION
25
ProteoSpin™ Detergent Clean-Up Maxi Kit
ProteoSpin™ Detergent Clean-Up Maxi Kit Components
•
•
•
•
•
•
•
Column Activation and Wash Buffer (Acidic)
Column Activation and Wash Buffer (Basic)
pH Binding Buffer (Acidic)
pH Binding Buffer (Basic)
Elution Buffer
Neutralizer
ProteoSpin™ SiC Maxi Spin Columns (filled with SiC) inserted
into 50 mL collection tubes
• Final Elution Tubes (50 mL)
• ProteoSpin™ Detergent Clean-Up Maxi Kit Application Manual
• ProteoSpin™ Detergent Clean-Up Maxi Kit Protocol Card
Recommended Storage Conditions
For unopened solution containers, the reagents should remain stable for 12
months when stored at room temperature. Once opened, all solutions,
except for the two binding buffers, should be stored at 4°C when not in use.
The binding buffers should remain at room temperature with the lids tightly
closed. Salt crystal formation may occur when stored at 4°C. If crystals
are visible, bring the entire bottle to room temperature and mix gently to
redissolve.
26
BIOTEK
CORPORATION
ProteoSpin™ Detergent Clean-Up Maxi Kit
Customer-Supplied Reagents and Equipment
You must have the following in order to use the ProteoSpin™ Detergent Clean-Up
Maxi Kit:
•
•
•
•
Micropipettors
• Isopropanol
pH Indicator paper
• Milli Q water
Other Elution Buffers (optional)
• 100 mL sterile bottles
Benchtop Centrifuge (capable of spinning the 50mL tubes)
Procedure
The ProteoSpin™ Detergent Clean-Up Maxi Kit is designed to remove detergents in solutions of acidic or basic proteins. Detergents are removed by phase partitioning with cation
exchange chromatography and the general principle for removal applies to different classes
of detergents.
The process applies to a wide variety of soluble proteins ranging from acidic to basic proteins. Two procedures, one for acidic proteins and another for basic proteins, are described.
In theory, the protocol for acidic proteins should apply to a majority of acidic and basic proteins since the resin is a cation exchanger; that is, all proteins with a pI greater than the binding pH at 4.5 should bind reversibly. However, basic proteins bind strongly when they are
used under these conditions, making their elution quite inefficient. Therefore, for soluble
basic proteins (pI ≥ 8), a different condition for binding the protein to the resin has been
developed. For the purposes of this kit, a protein whose pI is less than 8 will be treated as
acidic and will use the acidic protocol; otherwise, use the basic protocol.
BIOTEK
CORPORATION
27
ProteoSpin™ Detergent Clean-up Maxi Kit
Figure 2. Choosing a procedure based on the isoelectric point (pI)
Protocols
The following procedures describe the removal of detergents from acidic or basic proteins in
either buffered or unbuffered (dissolved in water) solutions. The kit utilizes spin columns
containing SiC, which bind the protein of interest if it retains a net positive charge. Nonspecifically bound proteins and detergents are washed from the column and the specific protein is eluted into specially formulated elution buffer, removing the detergent from the protein sample. Each spin column will remove detergent from up to 8 mg of acidic or basic protein. This kit is designed to remove a variety of detergents and/or surfactants from protein
solutions. It is capable of removing detergents such as Triton X-100, SDS, Chaps, NP-40 and
Tween 20. Each column has a capacity of up to 8 mg of protein, and will remove greater than
95% of detergents when present.
28
BIOTEK
CORPORATION
Detergent Clean-Up Protocol for Acidic Proteins
Detergent Clean-Up Protocol for Acidic Proteins
Proteins with isoelectric points (pI) of less than 7 are by definition acidic proteins. However,
for purposes of using the kit, the Protocol for Acidic Proteins applies to any protein whose pI
is less than 8.0. Proteins with pI higher than 8.0 are purified using the Protocol for Basic
Proteins. If the pI of the protein being purified is not known, the theoretical pI may be calculated using the web-based applications at http://us.expasy.org/tools/pi_tool.html or
http://www.up.univmrs.fr/~wabim/d_abim/compo-p.html.
Note: All centrifugation steps in these procedures are carried out at 1,000 x g in a benchtop
centrifuge. Please check your centrifuge’s user manual to determine the appropriate speed
that gives 1,000 x g. Performance of the kit is not affected by temperature, and thus the procedure may be performed at room temperature, 4ºC, or on ice. The user must take discretionary measures, such as chilling samples in ice, to preserve biological activity.
A. Sample Preparation
Note: In preparing your sample for column binding by centrifugation to removed detergents,
you will mix your protein solution with the pH Binding Buffer (Acidic) provided in the kit to
make proper adjustment of the pH. Isopropanol (not supplied with the kit) will also be added
to aid in the process. Make sure that all particulates in your sample have been removed by
either filtration or centrifugation. The column reservoir has a capacity for 20 mL hence
multiple centrifugations will be required for larger volumes.
BIOTEK
CORPORATION
29
Detergent Clean-Up Protocol for Acidic Proteins
1. Obtain your protein sample and determine the volume.
2. Depending on your starting protein solution, you will require a certain volume of pH
Binding Buffer (Acidic) to adjust the pH of your protein solution to 4.5. If the starting protein solution is in water, then add one part of the pH Binding Buffer (Acidic)
to 49 parts of the protein solution. However, if the starting protein solution is in a
buffer, you will need greater volumes of the pH Binding Buffer (Acidic) depending
on the buffer type and strength. Table 3 below serves only as guideline for the
amount of pH Binding Buffer (Acidic) to use for every milliliter volume of a protein
solution in a 100 mM buffer to obtain pH 4.5. Please check the pH after mixing and
add more if necessary to obtain the desired pH.
Please note that if the pH is at 4.5 or lower, do not add any binding buffer.
Table 3. pH Adjustment for Acidic Proteins
Starting pH of Solution
4
5
6
7
8
9
10
11
12
30
BIOTEK
CORPORATION
Volume of pH Binding Buffer (Acidic) per mL of protein solution (based on 100 mM buffered solution)
0 µL
20 µL
20 µL
20 µL
50 µL
80 µL
80 µL
80 µL
100 µL
Detergent Clean-Up Protocol for Acidic Proteins
3. Add one volume of isopropanol to the pH-adjusted solution.
4. Check the pH after mixing and add more pH Binding Buffer (Acidic) if necessary to
obtain the desired pH.
5. Set aside until the protein binding step.
B. Preparation of Modified Column Activation and Wash Buffer (Acidic)
This step prepares a modified solution to activate and wash the columns. It is prepared using
the provided pH Binding Buffer (Acidic) and isopropanol.
1. Combine the following in a sterile 100 mL bottle and mix well:
• 2 mL pH Binding Buffer (Acidic)
• 50 mL isopropanol
• 48 mL sterile deionized water
2. Label the container as Modified Column Activation and Wash Buffer (Acidic) and
keep it tightly sealed at room temperature when not in use. This volume is sufficient
for the entire Detergent Clean-up Maxi Kit.
BIOTEK
CORPORATION
31
Detergent Clean-Up Protocol for Acidic Proteins
C. Column Activation
1. Obtain a spin column with its 50 mL conical collection tube. Remove lid.
2. Apply 5 mL of Modified Column Activation and Wash Buffer (Acidic) to the column. Screw the cap back on LOOSELY.
3. Centrifuge for two minutes at 1,000 x g.
Note: Start timing only after centrifuge has reached desired speed.
4. Repeat steps 2 and 3 to complete the column activation step. Discard flowthrough.
D. Protein Binding
During the binding step, the protein solution is passed through the resin bed aided by centrifugation. The protein is captured as it comes in contact with the resin. For most protein samples
tested, the centrifugation time provides sufficient contact time for the protein to bind.
1. Apply the protein sample (from the Sample Preparation step) onto the column and
centrifuge for two minutes. A maximum of 20 mL can be added at one time.
2. Discard the flowthrough. Reassemble the spin column with its collection tube.
Note: You can save the flowthrough in a fresh tube for assessing your protein's
binding efficiency.
32
BIOTEK
CORPORATION
Detergent Clean-Up Protocol for Acidic Proteins
3. Depending on your sample volume, repeat steps 1 and 2 until the entire protein
sample has been applied to the column.
4. Discard any remaining flowthrough and reassemble the spin column with its
collection tube.
E. Column Wash
This step removes non-specifically bound debris from the column.
1. Apply 10 mL of Modified Column Activation and Wash Buffer (Acidic) to the column and centrifuge for two minutes.
2. Discard the flowthrough and reassemble the spin column with its collection tube.
3. Add 10 mL of Regular Column Activation and Wash Buffer (Acidic) to the column
and centrifuge for two minutes.
4. Inspect the column and ensure that the liquid has passed through into the collection
tube. There should be no liquid in the column. If necessary, spin an additional two
minutes to dry.
BIOTEK
CORPORATION
33
Detergent Clean-Up Protocol for Acidic Proteins
F. Protein Elution
The Elution Buffer that is supplied is 50 mM sodium phosphate pH 12.5. Please refer to
Appendix 1 (Optional Elution Buffers) for a list of alternate elution solutions that have been
tested with the kit. The proteins in the buffer should be neutralized immediately. It is therefore recommended that the Neutralizer that is provided is pre-added to the elution tube (fresh
50 mL conical tube). However, this is not needed for the first elution. For the second elution,
0.3 mL of Neutralizer is needed. Note: Please verify the pH of your first eluted protein sample
and adjust with the Neutralizer if required.
1. Add 300 µL Neutralizer, if desired (not required for the 1st elution), to fresh 50 mL
collection tube.
2. Transfer the spin column, with bound protein, into the 50 mL collection tube from
step 1.
3. Apply 4 mL of Elution Buffer to column and centrifuge to elute bound protein.
4. A second elution is optional (another 4 mL). Repeat Steps 1 to 3 if desired. Keep
separate from the 1st elution.
Note: Approximately 90% of bound protein is recovered in the first two elutions. If desired, a
third elution using Elution Buffer may be carried out. This should be collected into a different
tube (to which the Neutralizer is pre-added) to prevent dilution of the first two elutions.
Protein samples are now ready for downstream applications.
34
BIOTEK
CORPORATION
Detergent Clean-Up Protocol for Basic Proteins
Detergent Clean-Up Protocol for Basic Proteins
Proteins with isoelectric points (pI) of less than 7 are by definition acidic proteins. However,
for purposes of using the kit, the Protocol for Acidic Proteins applies to any protein whose pI is
less than 8.0. Proteins with pI higher than 8.0 are purified using the Protocol for Basic
Proteins. If the pI of the protein being purified is not known, the theoretical pI may be calculated using the web-based applications at http://us.expasy.org/tools/pi_tool.html or
http://www.up.univmrs.fr/~wabim/d_abim/compo-p.html.
Note: All centrifugation steps in these procedures are carried out at 1,000 x g in a benchtop
centrifuge. Please check your centrifuge’s user manual to determine the appropriate speed that
gives 1,000 x g. Performance of the kit is not affected by temperature, and thus the procedure
may be performed at room temperature, 4ºC, or on ice. The user must take discretionary measures, such as chilling samples in ice, to preserve biological activity.
A. Sample Preparation
Note: In preparing your sample for column binding by centrifugation to remove detergents,
you will mix your protein solution with the pH Binding Buffer (Basic) provided in the kit to
make proper adjustment of the pH. Isopropanol (not supplied with the kit) will also be added to
aid in the process. Make sure that all particulates in your sample have been removed by either
filtration or centrifugation. The column reservoir has a capacity for 20 mL hence multiple centrifugations will be required for larger volumes.
BIOTEK
CORPORATION
35
Detergent Clean-Up Protocol for Basic Proteins
1. Obtain your protein sample and determine its volume.
2. Depending on your starting protein solution, you will require a certain volume of the
pH Binding Buffer (Basic) to adjust the pH of your protein solution to 7.0. For
example, if the starting protein solution is in water, add one (1) part of the pH
Binding Buffer (Basic) to 49 parts of the protein solution. If the starting protein solution is in a buffer, you may need greater volumes of the pH Binding Buffer (Basic) to
lower the pH, depending on the buffer type and its ionic strength. Table 4 below
serves only as guideline for the amount of pH Binding Buffer (Basic) to use for
every milliliter volume of a protein solution in a 100 mM buffer to obtain pH 7.0.
Please check the pH after mixing and add more if necessary to obtain the desired pH.
Table 4. pH Adjustment for Basic Proteins
Starting pH of Solution
4
5
6
7
8
9
10
11
12
36
BIOTEK
CORPORATION
Volume of pH Binding Buffer (Basic) per mL of protein solution (based on 100 mM buffered solution)
150 µL
80 µL
80 µL
0 µL
60 µL
60 µL
60 µL
80 µL
80 µL
Detergent Clean-Up Protocol for Basic Proteins
3. Add one volume of isopropanol to the pH-adjusted solution.
4. Mix contents well and measure the pH before applying to the column. Further adjust
the pH if necessary.
5. Set aside until the protein binding step.
B. Preparation of Modified Column Activation and Wash Buffer (Basic)
This step prepares a modified solution to activate and wash the columns. It is prepared using
the provided pH Binding Buffer (Basic) and isopropanol.
1. Combine the following in a sterile 100 mL bottle and mix well:
• 2 mL of pH Binding Buffer (Basic)
• 50 mL of isopropanol
• 48 mL of sterile deionized water
2. Label the container as Modified Column Activation and Wash Buffer (Basic) and
keep it tightly sealed at room temperature when not in use. This volume is sufficient
for the entire Detergent Clean-Up Maxi Kit.
BIOTEK
CORPORATION
37
Detergent Clean-Up Protocol for Basic Proteins
C. Column Activation
1. Obtain a spin column with its 50 mL conical collection tube. Unscrew the cap.
2. Apply 5 mL of Modified Column Activation and Wash Buffer (Basic) to the column.
Screw the cap back on LOOSELY.
3. Centrifuge for two minutes at 1,000 x g.
Note: Start timing only after centrifuge has reached desired speed.
4. Repeat steps 2 and 3 to complete the column activation step. Discard the flowthrough.
38
BIOTEK
CORPORATION
Detergent Clean-Up Protocol for Basic Proteins
D. Protein Binding
During the binding step, the protein solution is passed through the resin bed aided by centrifugation. The protein is captured as it comes in contact with the resin. For most protein samples
tested, the centrifugation time provides sufficient contact time for the protein to bind.
1. Apply the protein sample (from the Sample Preparation step) onto the column
and centrifuge for two minutes. A maximum of 20 mL can be added at one time.
2. Discard the flowthrough. Reassemble the spin column with its collection tube.
Note: You can save the flowthrough in a fresh tube for assessing your protein's
binding efficiency.
3. Depending on your sample volume, repeat steps 1 and 2 until the entire protein
sample has been applied to the column.
4. Discard any remaining flowthrough and reassemble the spin column with its
collection tube.
BIOTEK
CORPORATION
39
Detergent Clean-Up Protocol for Basic Proteins
E. Column Wash
This step removes non-specifically bound debris from the column.
1. Apply 10 mL of Modified Column Activation and Wash Buffer (Basic) to the
column and centrifuge for two minutes.
2. Discard the flowthrough and reassemble the spin column with its collection
tube.
3. Add 10 mL of Regular Column Activation and Wash Buffer (Basic) to the column and centrifuge for two minutes.
4. Inspect the column and ensure that the liquid has passed through into the collection tube. There should be no liquid in the column. If necessary, spin an additional two minutes to dry.
F. Protein Elution
The Elution Buffer that is supplied is 50 mM sodium phosphate pH 12.5. It is recommended
that upon elution, the protein solution is neutralized immediately especially for proteins that
are known to be sensitive to high pH. The following steps are suggested if the supplied
Elution Buffer is used and neutralization is desired. For a list of alternate elution solutions that
have been tested for the kit, please refer to Appendix 1 (Optional Elution Buffers).
40
BIOTEK
CORPORATION
1. Add 300 µL of Neutralizer to a fresh 50 mL collection tube (supplied)
2. Transfer the spin column, with bound protein, into the 50 mL collection tube from
step 1.
3. Apply 4 mL of Elution Buffer to column and centrifuge to elute bound protein.
4. A second elution is optional (another 4 mL). Repeat steps 1 to 3 if desired.
Keep separate from the 1st elution.
Note: Approximately 90% of bound protein is recovered in the first two elutions. If
desired, a third elution using Elution Buffer may be carried out. This should be collected into a different tube (to which Neutralizer is pre-added) to prevent
dilution of the first two elutions.
Protein samples are now ready for downstream applications.
BIOTEK
CORPORATION
41
A
Appendix 1.
Optional Elution Buffers
Proteins bound to SiC via interactions with electrostatic charges are eluted through pHdependent mechanisms. The efficiency of protein elution depends on high pH above the pI of
the protein to be purified. The pH of the elution buffer chosen must be at least one unit higher
than the pI (isoelectric point) of the protein of interest. Solutions not provided with the
ProteoSpin™ Maxi CBED or Detergent Clean-Up Maxi kits may be utilized if they are more
appropriate for your needs. The table below describes optional elution buffers and their
observed efficiency when BSA is used as a test protein.
Table 5. Alternate Elution Buffers
Elution Buffers
50 mM ammonium hydroxide (approximate pH 11)
250 mM ammonium hydroxide (approximate pH 11)
1 M ammonium hydroxide (approximate pH 11)
1 M ethanolamine (approximate pH 9)
50 mM sodium phosphate (approximate pH 12.5)
500 mM sodium phosphate (approximate pH 12.5)
100 mM sodium borate (approximate pH 12.5)
1 M Tris (approximate pH 12.5)
42
BIOTEK
CORPORATION
Approximate Protein Recovery
70%
70%
90%
70-80%
95%
<70%
95-100%
95%
B
Appendix 2.
Proteins with Established Isoelectric Points
Table 6. Examples of Proteins with Known Isoelectric Points
Protein
Albumin, bovine serum
Albumin, human serum
Carbonic anhydrase
Carboxypeptidase
Catalase
Cytochrome C
Fibrinogen
Growth hormone, human
Hemoglobin, horse
Immunoglobulin G
Insulin
Lysozyme, hen egg white
Myoglobin, horse
Ovalbumin
Pepsin
Ribonuclease
Thyroglobulin
Trypsin inhibitor, soybean
Urease
Molecular Weight (kDa)
67
66.5
30
34
250
13
330
21.5
65
150
5.7
14.3
17
40
35.5
14
660
22.5
480
Iso-Electric Point (pI)
5.5
4.8
7.3
6.0
5.6
10.6
5.5
6.9
6.9
6.4-7.2
5.3
11.0
7.0
4.6
< 1.0
7.8
4.6
4.55
5.1
BIOTEK
CORPORATION
43
Appendix 3.
Frequently Asked Questions and Troubleshooting
Question
CBED Detergent
Removal
What is the dark gray
material that is packed
into the spin columns?
•
How does the kit
remove detergents?
How does the kit concentrate and desalt
proteins?
44
BIOTEK
•
CORPORATION
C
Answer
•
The dark gray chromatography material is silicon carbide (SiC).
It is processed using proprietary non-chemical methods to function as an ion exchange chromatography resin.
•
The kit removes detergents from aqueous solutions by taking
advantage of the poor affinity of detergents to silicon carbide,
the chromatographic resin used. The resin works in conjunction with isopropanol to eliminate detergents including those
that bind to proteins such as SDS. Please refer to the application note “Efficient Removal of SDS from Proteins Solutions”
on our website at http.//www.norgenbiotek.com.
The resin has high affinity for proteins with net positive
charges, but has very poor affinity for monovalent and divalent
cations. When dilute protein solutions with high salt pass
through the resin, the charged proteins are captured by the resin
while the aqueous solution with the salts is removed as the
flowthrough. The bound protein can be recovered by eluting it
in small volumes and in salt-free buffers to achieve several-fold
concentration.
Frequently Asked Questions and Troubleshooting
Question
What proteins can
be processed using
this kit?
CBED Detergent
Removal
•
•
Answer
All soluble (acidic and basic) proteins can be processed
using this kit.
What can I do if I do
not know the isoelectric point (pI) of my
protein of interest?
•
•
The isoelectric point of a protein (pl) may be theoretically computed from its amino acid composition through the aid of webbased applications such as
http://us.expasy.org/tools/pi_tool.html. Another site at
http://www.up.univ-mrs.fr/~wabim/d_abim/compo-p.html
determines isoelectric points (pl) with considerations for position of residues in the polypeptide chain. The calculated pl
should help determine whether the acidic or basic protocol is to
be followed. Alternatively, if there is sufficient protein sample,
the protein can be bound and eluted using first the acidic protocol, and then the basic protocol, and the recovery compared to
determine which protocol gives better results.
Can the columns
be re-used?
•
•
Columns are designed for single use only. If used more than
once, sample carry-over may cause cross-contamination.
How should I store
the kit solutions?
•
Once opened, the solutions in their containers should be stored
at 4 °C when not in use, except the pH Binding Buffers, which
remains at room temperature with the lids tightly closed. If any
precipitation of the reagents occurs, warm to room temperature
and dissolve the crystals prior to use. If unopened, the solution
containers may be stored at room temperature.
BIOTEK
CORPORATION
45
Frequently Asked Questions and Troubleshooting
Question
CBED Detergent
Removal
Do I need to pre-filter my
sample before loading it
onto the column?
How should I prepare
my sample before loading it onto the column?
•
•
•
Answer
Insoluble materials, if present in the protein solution, should be
spun down and the clarified solution applied to the column.
•
Ensure that the pH of the sample is the same as the pH of the
Column Activation and Wash Solution that you are using.
Also, ensure that the amount of protein in your sample will not
overload the column capacity (8 mg).
What are the maximum and minimum
sample volumes that I
can load onto the column?
•
•
We recommend that no more than 20 mL of sample be loaded
onto the column, to ensure that the tip of the column does not
rest in the flowthrough that is caught in the collection tube.
Excess volumes can be loaded in subsequent spins as long as
the collection tube is emptied between loads. The minimum
load should be at least 4 mL to completely cover the resin bed.
What are the maximum
and minimum amounts of
protein that I can load
onto the column?
•
•
We recommend between 250 µg and 8 mg of sample be loaded
onto the column in order to obtain high percentage recovery.
•
We recommend a speed around 1,000 x g in the benchtop centrifuge. Speeds of more than 1,500 x g should be avoided.
Lower speeds can be investigated to maximize efficiency.
Additional spinning times may be required to ensure all the liquid has passes through.
What are the highest
and lowest recommended microcentrifuge speeds for the
columns?
46
BIOTEK
•
CORPORATION
Frequently Asked Questions and Troubleshooting
Question
What is the typical
amount of protein recovered that I can expect
with the ProteoSpin™
columns?
Can I autoclave the
columns?
CBED Detergent
Removal
•
•
Recovery of BSA in water was found to be greater than 90%
when testing 8 mg of protein. Recovery depends on the specific
protein sample, and varies with detergent concentrations. If
detergents are present, use the Detergent Clean-Up Kit.
•
•
No. The columns are not designed to be autoclaved as it causes
the plastic in the columns to deform.
•
Yes, the kit is capable of removing all types of detergents
including anionic, cationic, zwitterionic and nonionic. SDS can
be removed efficiently with the kit, including tightly bound
SDS complexed with proteins.
Can all types of detergents be removed
using this kit? What
about SDS?
Do any detergents or
reagents interfere with
the performance of the
columns?
How can I tell if my
protein bound correctly to the column?
Answer
•
Most commonly used biological detergents have little or no
effect on column performance except for SDS. Also, greater
than 1M concentration of urea and guanidine salts are known to
interfere with protein binding. For the removal of detergents,
use the ProteoSpin Detergent Clean-Up Kit.
•
Save the flowthrough from the binding step and perform a protein assay to determine the amount of protein. To determine the
amount of protein present, use a reliable colorimetric assay.
Several are commercially available. To eliminate inaccuracies,
make sure that the protein standard used is in the same buffer
condition as the flowthrough.
•
BIOTEK
CORPORATION
47
Frequently Asked Questions and Troubleshooting
Question
CBED Detergent
Removal
Answer
I used the recommended binding pH, but
still my protein did
not bind correctly.
Can I use a different
binding pH?
•
•
Yes. The binding pH may be adjusted depending on the pI of
your protein. It is suggested to start with a pH that is one unit
lower than the pI, and if necessary, lowering the pH from there.
If the provided pH binding buffer is used, it can be adjusted to
a pH as low as 3.7 by titrating it with HCl or glacial acetic acid.
For proteins with an even lower pI, the pH binding buffer may
be substituted with a 500 mM phosphate buffer (prepared by
mixing 9 parts of 500 mM phosphoric acid with 1 part 500 mM
trisodium phosphate) and used as a 10-fold concentrate.
Am I able to check the
activity of my protein
by using the eluted
protein directly?
•
•
If the elution buffer provided in the kit was used, then the eluted protein may be used directly to check activity of the protein.
The elution buffer is sodium phosphate and is compatible with
many assays for biological activity.
I use a colorimetric
protein assay to determine concentration of
eluted proteins. Is
there an accurate way
of doing it?
48
BIOTEK
•
CORPORATION
•
The colorimetric protein assays available in the market are
quite accurate, however, there are a number of ways to improve
them. First, using a linear regression to fit the points for the
standard curve is often less accurate than using a second-degree
polynomial. Microsoft Excel™ has full features for curve fitting. The goodness-of-fit for the regression should determine
whether first-degree (linear) or second-degree (polynomial)
would be used. Second, the protein used as the standard should
closely match the dye-binding characteristics of the protein of
interest. For example, using BSA as a standard to determine
concentrations of immunoglobulins may provide erroneous
results. This is because immunoglobulins do not bind some
dyes efficiently. Third, ensure that the protein standard is in a
solution that resembles the solution of the test protein.
Sometimes buffer constituents can have drastic effects on dye
binding properties.
Frequently Asked Questions and Troubleshooting
Question
The acidic and basic
methods are almost
identical, except for
the solutions used.
Why is it necessary to
separate the two?
The binding pH is 4.5,
yet my protein has a pI
of 4. Do I need to
adjust the pH of my
binding solution to
lower than 4.5? If so,
how would I do that?
CBED Detergent
Removal
•
•
•
•
Answer
In ion exchange chromatography, it is customary to use either a
cation or anion exchanger resin, depending on the type of target
protein. For SiC resin, this carries negative charges on its surface, proteins bind through positive charges on their side chains
that are exposed on the surface. In theory, the acidic method
should suffice to capture all basic and acidic proteins. In practice, however, we find that basic proteins bound under acidic
conditions are difficult to elute, and therefore decrease the
overall performance of the columns. Binding basic proteins at
pH 7 reduces column affinity for such proteins, making elution
more efficient.
If your protein has a pI lower than 4.5, it is almost always necessary to lower the pH of the protein solution so that it is one
pH unit lower than the protein's pI. If the ProteoSpin™ pH
Binding Buffer for Acidic Proteins is used, the final pH may be
adjusted to as low as 3.75 by titrating with 100 mM HCl. If a
lower binding pH is desired, you may use different buffers,
such as phosphate (pKa1 = 2.15). Many acidic proteins tend to
precipitate as the pH of the solution approaches their pI. If protein precipitation is apparent (solution becomes cloudy), diluting the solution to a lower protein concentration may solve the
problem. Using a binding pH of one-half unit below the protein's pI is also feasible, although the expected recovery for the
bound protein will be lower.
BIOTEK
CORPORATION
49
Frequently Asked Questions and Troubleshooting
Question
CBED Detergent
Removal
When desalting my
basic protein, my
protein yield is
generally poor.
Why is this and
what is the best way
to overcome the
problem?
How can I separate
an acidic protein from
a basic one when both
are present in the same
solution?
50
BIOTEK
Basic proteins generally are more difficult to recover from ion
exchange columns than acidic proteins. The problem usually
lies in basic proteins' high affinity to negatively charged ion
exchange resins. Elution of these proteins may require a combination of high pH and high ionic strength (by adding salt) in the
elution buffer. Although SiC has low affinity for salts, which
allows it to be an effective desalter, high salts can affect proteins severely. For basic proteins, the effect is reduction of
binding efficiency. To overcome the problem, dilute the basic
protein solution so that the salt concentration is 0.2 M or lower,
then use the kit to concentrate the protein and desalt at the same
time. Note that recovery of the basic protein increases with
decreasing salt concentration achieved by dilution. Fortunately,
the ability of acidic proteins to bind does not seem to be affected by high salt concentrations, hence the normal protocol may
be used without modification.
•
•
CORPORATION
Answer
•
Separation of proteins using SiC ion exchange chromatography
is achieved by differential binding of proteins due to their pI.
When separating an acidic protein from a mixture with a basic
protein, use the Basic Protocol.
Frequently Asked Questions and Troubleshooting
Question
CBED Detergent
Removal
Can I purify a protein
in a glycerol-containing solution?
•
Can I purify a protein
in a sugar-containing
solution?
•
Answer
Glycerol of less than 20% concentration has little effect on the
binding of most proteins to the column. At higher glycerol
concentrations (as high as 60% v/v) , the recovery rate may be
reduced by as much as 50%. Note that the ProteoSpin™
Detergent Clean-Up Kit must not be used with proteins that
contain glycerol.
•
Sucrose has very little effect, even at as high a
concentration as 1M.
BIOTEK
CORPORATION
51
D
Appendix 4.
Troubleshooting Guide
Each of the following tables discusses a problem which can occur, its possible
causes, and presents solutions and explanations.
Problem: Protein Solution Does Not Flow Through the Column
Possible Causes
Centrifugation speed was too low.
Inadequate spin time
Protein solution is too viscous
Cellular debris is present in the protein solution
Protein solution is not completely dissolved
52
BIOTEK
CORPORATION
g
Solution and Explanation
Check the centrifuge and ensure it is capable
of generating 1,000 x g. Sufficient centrifugal
force is required to push the liquid through the
column.
Spin an additional minute or two to ensure the
liquid is through the column.
Dilute the protein solution and adjust the pH to
either pH 4.5 or 7. Highly viscous materials due
to high protein concentrations can slow down
flow rate significantly.
Filter the sample with a 0.45 M filter or spin
down insoluble materials and transfer liquid
portion to the column.
Solid, insoluble
materials can cause severe clogging problems.
Dissolve the sample in a larger amount of
buffer. Solid, insoluble materials can cause
clogging problems.
Troubleshooting Guide
Problem: Poor Protein Recovery
y
Possible Causes
Incorrect procedure was used.
Initial volume of sample applied to the column
was too low.
Incorrect pH adjustment of samples.
Protein may have precipitated prior to loading
onto the column.
Solution and Explanation
Ensure that the acidic protocol was used for
acidic proteins and the basic protocol was used
for basic proteins. It is known that when basic
proteins are bound with the acidic protocol,
elution is inefficient because the basic proteins
are bound too tightly.
Load at least 4 mL onto the column. This
volume ensures that the entire bed is covered
sufficiently.
Ensure that the pH of the sample is 4.5 for
acidic proteins and pH 7.0 for basic proteins.
If the pH of the protein solution is the same as
the pI of the protein, precipitation may occur.
In this case, adjust the pH of the sample to at
least one pH lower than the pI of your protein.
Problem: Eluted Protein is Degraded
g
Possible Causes
Eluted protein was not neutralized.
Eluted protein solution was not neutralized
quickly enough.
Proteases may be present.
Bacterial contamination of the protein solution.
Solution and Explanation
Add 300 L of Neutralizer to each 4mL of
eluted protein for all basic protein elutions.
Acidic proteins tend not to need the
neutralization for the first elution but require it
for the second or third. Check the pH of the
first elution if you are using an acidic protein.
If eluted protein is not neutralized immediately,
degradation will occur.
We strongly
recommend adding Neutralizer in order to
lower the pH.
Use protease inhibitors during all steps of the
Sample Preparation.
Prepare the protein sample with 0.015%
sodium azide. The elution buffer already
contains sodium azide.
BIOTEK
CORPORATION
53
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement