Bio-Rad ProteoMiner Protein Enrichment Kits User Manual
ProteoMiner
™
Protein
Enrichment Kits
Instruction Manual
Catalog #
163-3003
163-3006
163-3007
163-3008
163-3009
163-3010
163-3011
163-3012
For Technical Support, contact your local Bio-Rad office, or
in the US, call 1-800-4BIORAD (1-800-424-6723)
Table of Contents
Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Section 2 Kit Specifications . . . . . . . . . . . . . . . . . . . . . . . .1
Section 3 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . .3
Section 4 Reagent Preparation . . . . . . . . . . . . . . . . . . . . .4
Section 5 Sample Considerations . . . . . . . . . . . . . . . . . . . . . .4
Section 6 Instructions for Use With ProteoMiner™
Large-Capacity Kits
(Catalog #s 163-3007 and 163-3009) . . . . . . . . .4
Section 7 Instructions for Use With ProteoMiner
Small-Capacity Kits
(Catalog #s 163-3006 and 163-3008) . . . . . . . . .7
Section 8 Instructions for Use With Sequential
Elution Large-Capacity Kit
(Catalog #163-3011) . . . . . . . . . . . . . . . . . . . . . .9
Section 9 Instructions for Use With Sequential
Elution Small-Capacity Kit
(Catalog #163-3010) . . . . . . . . . . . . . . . . . . . . .12
Section 10 Instructions for Use With Bulk Beads
(Catalog #163-3012) . . . . . . . . . . . . . . . . . . . . .15
Section 11 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Section 12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Section 13 Product Information . . . . . . . . . . . . . . . . . . . . .18
Section 1
Introduction
The ProteoMiner™ technology is a novel sample preparation tool used for the
compression of the dynamic range of the protein concentration in complex
biological samples. High-abundance proteins present in complex biological
samples like sera or plasma, make the detection of medium- and lowabundance proteins extremely challenging. This technology provides a method
of overcoming this challenge, allowing for the exploration of the entire
proteome.
This is accomplished through the use of a large, highly diverse bead-based
library of combinatorial peptide ligands. When complex biological samples are
applied to the beads, the high-abundance proteins saturate their high affinity
ligands and excess protein is washed away. In contrast, the medium- and lowabundance proteins are concentrated on their specific affinity ligands. This
reduces the dynamic range of protein concentrations while maintaining
representatives of all proteins within the original sample.
Section 2
Kit Specifications
ProteoMiner™ Small-Capacity Kit (Catalog #163-3006)
Provides reagents for processing 10 samples. Compatible with 2-D gel
electrophoresis and other downstream protein separation analysis methods.
• Spin Columns. 10 columns each containing 500 µl bead slurry (4% beads,
20% v/v aqueous EtOH), 20 µl settled bead volume
• Wash Buffer. 50 ml PBS buffer (150 mM NaCl, 10 mM NaH
2PO4
, pH 7.4)
• Elution Reagent. 2 vials, lyophilized urea CHAPS (8 M urea, 2% CHAPS)
• Rehydration Reagent. 5 ml, 5% acetic acid
• Collection Tubes. 20 capless, 2 ml centrifuge tubes; 10 capped, 2 ml
centrifuge tubes
• Instruction Manual
• Quick Start Guide
ProteoMiner Large-Capacity Kit (Catalog #163-3007)
Provides reagents for processing 10 samples. Compatible with 2-D gel
electrophoresis and other downstream protein separation analysis methods.
• Spin Columns. 10 columns each containing 500 µl bead slurry (20%
beads, 20% v/v aqueous EtOH), 100 µl settled bead volume
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• Wash Buffer. 50 ml PBS buffer (150 mM NaCl, 10 mM NaH2PO4, pH 7.4)
• Elution Reagent. 5 vials, lyophilized urea CHAPS (8 M urea, 2% CHAPS)
• Rehydration Reagent. 5 ml, 5% acetic acid
• Collection Tubes. 20 capless, 2 ml centrifuge tubes; 10 capped, 2 ml
centrifuge tubes
• Instruction Manual
• Quick Start Guide
ProteoMiner Introductory Small-Capacity Kit (Catalog #163-3008)
Contains two spin columns, all other reagents are the same as in the
ProteoMiner small-capacity kit above. Introductory kit provides reagents for
processing two samples.
ProteoMiner Introductory Large-Capacity Kit (Catalog #163-3009)
Contains two spin columns, all other reagents are the same as in the
ProteoMiner large-capacity kit above. Introductory kit provides reagents for
processing two samples.
ProteoMiner Sequential Elution Small-Capacity Kit (Catalog
#163-3010)
This kit combines the ProteoMiner small-capacity kit (catalog #163-3006) and
the ProteoMiner sequential elution reagents (catalog #163-3003) and is
designed to provide multiple elution options for researchers using SELDI or
other downstream protein separation analysis methods, and who wish to
access additional proteins. This kit is NOT compatible with 2-D gel
electrophoresis.
ProteoMiner Sequential Elution Large-Capacity Kit (Catalog
#163-3011)
` This kit combines the ProteoMiner large-capacity kit (catalog #163-3007) and
the ProteoMiner sequential elution reagents (catalog #163-3003) and is
designed to provide multiple elution options for researchers using SELDI or
other downstream protein separation analysis methods, and who wish to
access additional proteins. This kit is NOT compatible with 2-D gel
electrophoresis.
ProteoMiner Sequential Elution Reagents (Catalog #163-3003)
To be used in combination with the ProteoMiner kits (catalog #s163-3006 or
163-3007), this product provides reagents for processing 10 samples. The
sequential elution reagents are available for researchers using SELDI or other
downstream protein separation analysis methods, and who wish to access
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additional proteins. These reagents are NOT compatible with 2-D gel
electrophoresis.
• Elution Reagent 1. 5 ml 1 M sodium chloride, 20 mM HEPES, pH 7.4
• Elution Reagent 2. 5 ml 200 mM glycine, pH 2.4
• Elution Reagent 3. 5 ml 60% ethylene glycol in water
• Elution Reagent 4. 5 ml 33.3% 2-propanol, 16.7% acetonitrile, 0.1%
trifluoroacetic acid
• Plasma Preparation Buffer. 1.5 ml 1 M sodium citrate, 20 mM HEPES,
pH 7.4
• Collection tubes. 30 capped, 2 ml centrifuge tubes
Items required but not provided:
• Microcentrifuge or vacuum manifold (available through Bio-Rad, catalog
#732-6470)
• Pipet
• Proteomics grade water (available through Bio-Rad, catalog #163-2091)
ProteoMiner Beads (Catalog #163-3012)
Provides 525 mg of dry beads, columns and reagents not provided.
Section 3
Storage Conditions
Store the unopened kit at 4°C. After reconstituting lyophilized elution reagent
store any remaining material at –20°C for up to one week.
3
Section 4
Reagent Preparation
Prepare elution reagent by adding 610 µl rehydration reagent to one vial
lyophilized elution reagent. Following rehydration, each vial will contain enough
elution reagent for processing two samples. If preparing an uneven number of
columns, you will have remaining material that will be required for subsequent
preparations. Remaining material may be stored at –20°C for up to one week.
However, it is recommended to make this solution fresh each time you use the
kit.
Section 5
Sample Considerations
This kit has been optimized for plasma (nonheparinized) and serum samples.
Sample loading and buffer compatibility have not been validated for other
sample types. However, ProteoMiner™ technology has successfully been
applied to other sample types including urine (Righetti et al. 2005), bile
(Housset et al. 2007), platelets (Boschetti et al. 2008), red blood cell extract
(Boschetti et al. 2008), and egg white extract (Boschetti et al. 2008).
Results with other sample types will vary depending on the amount of protein
in your sample; best results are obtained with 50 mg protein for the large-capacity
kits and 10 mg for the small-capacity kits. Caution: When working with
human plasma/serum it is important to follow biohazardous
material handling guidelines.
The ratio of protein to beads is crucial for optimal performance of ProteoMiner
kits. The dynamic range of the protein concentration in the sample is reduced
when the high-abundant proteins saturate their ligands and the low-abundant
proteins bind to a sufficient number of ligands to allow for enrichment.
Therefore, in order to achieve optimal results, it is important to load the
recommended amount of protein.
Section 6
Instructions for Use With ProteoMiner™
Large-Capacity Kits (Catalog #s 163-3007
and 163-3009)
This protocol has been optimized for plasma and serum samples with
protein concentrations of >
50 mg/ml (requires total protein load >50 mg). For
other sample types, please refer to Section 5: Sample
Considerations.
4
Note: A ProteoMiner sequential elution kit (catalog #163-3011) is available for
researchers using SELDI or other downstream protein separation analysis
methods, other than 2-D gel electrophoresis, and who wish to access
additional proteins.
If using the ProteoMiner sequential elution kit, refer to page 9.
Step 1 – Column Preparation
Vacuum (at 16 mm Hg) can replace centrifugation for column preparation,
sample binding and sample wash steps if desired. (Vacuum manifold is
available through Bio-Rad, catalog #732-6470.)
1. First remove the top cap and then snap off the bottom cap from each of
the spin columns you will be using.
Note: Do not discard top or bottom caps, they will be reused throughout
the protocol. If beads settle in top cap, replace after removing bottom
plug and centrifuge with top cap on column. To use bottom cap as a
plug, invert and firmly place in bottom of spin column.
2. Place the column in a capless collection tube and centrifuge at 1,000 x g
for 30–60 sec to remove the storage solution. Discard collected material.
Note: Kit contains one capless collection tube per spin column for the
following steps: column preparation, sample binding, and sample wash.
Kit contains one capped collection tube per spin column to be used for
the elution step, allowing for easy storage of your eluted sample.
3. Replace the bottom cap and add 600 µl wash buffer, then replace top cap.
4. Rotate column end-to-end several times over a 5 min period.
5. Remove bottom cap, place the column in a capless collection tube and
centrifuge at 1,000 x g for 30–60 sec to remove buffer. Discard collected
material.
6. Repeat steps 3 and 4.
7. Remove caps, place the column in a capless collection tube and
centrifuge at 1,000 x g for 30–60 sec to remove the wash buffer. Discard
collected material.
8. Replace bottom cap on spin column. The column now contains 100 µl of
settled beads and is ready for sample binding.
Step 2 – Sample Binding
Samples should be free of precipitate. If needed, centrifuge samples at
10,000 x g for 10 min to clarify. Take precautions to avoid the bottom
aggregate proteins and top lipid layer when recovering your sample. It is
recommended that at least 1 ml of sample (protein concentration
≥50 mg/ml) is
added to the column, as lower volumes may not achieve optimal results. For
other sample types, please refer to Section 5: Sample
Considerations.
1. Add 1 ml of sample to column. Replace top cap and rotate column on a
platform or rotational shaker for 2 hr at room temperature.
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