0010636D:4110137B-4.0.GR.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page TOC1 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 . . . . . . . . . . . . . . . . . . . . . .
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 1 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.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 2 • 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.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 3 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.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 4 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.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 5 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.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 6 Note: If using plasma, clumping may occur after 1 hr of binding; this is expected and will not negatively impact your sample preparation. Heparinized plasma is not compatible with this kit. Step 3 – Sample Wash 1. Remove bottom cap, place column in a capless collection tube and centrifuge at 1,000 x g for 30–60 sec. Discard collected material. 2. Replace the bottom cap and add 600 µl of wash buffer to column.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 7 Section 7 Instructions for Use With ProteoMiner™ Small-Capacity Kits (Catalog #s 163-3006 and 163-3008) This protocol has been optimized for plasma and serum samples with protein concentrations of >50 mg/ml (requires total protein load >10 mg). For other sample types, please refer to Section 5: Sample Considerations.
010636D:4110137B-4.0.GR.qxd 8. 3/12/2009 7:51 AM Page 8 Replace bottom cap on spin column. The column now contains 20 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.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 9 5. Incubate column at room temperature, lightly vortex several times over a period of 15 min. 6. Remove caps, place in a clean collection tube labeled E1 and centrifuge at 1,000 x g for 30–60 sec. This elution contains your eluted proteins. Do not discard. 7. Repeat steps 5–6 two more times. (Elutions may be pooled or analyzed individually. If analyzed individually, you will need additional collection tubes not provided with kit.) 8.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 10 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.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 11 2. Replace the bottom cap and add 600 µl of wash buffer to column, then replace top cap and rotate end-to-end several times over a 5 min period. 3. Remove bottom cap, place column in a capless collection tube and centrifuge at 1,000 x g for 30–60 sec. Discard collected material. 4. Repeat steps 2 and 3 three more times.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 12 11. Repeat steps 9 and 10 two more times and collect both elutions in tube F3. 12. Attach bottom cap to column and add 100 µl of elution reagent 4 to column. Incubate at room temperature and lightly vortex several times over a period of 5 min. 13. Remove bottom cap, place column in collection tube labeled F4 and centrifuge at 1,000 x g for 30–60 sec to collect the elution. This elution contains your eluted proteins. Do not discard. 14.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 13 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 200 µl wash buffer, then replace top cap. 4. Rotate column end-to-end several times over a 5 min period. 5.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 14 centrifuge at 1,000 x g for 30–60 sec. Discard collected material. 4. Repeat steps 2 and 3 two more times. Step 4 – Sequential Elution (Reagents required for this step are included with the ProteoMiner sequential elution reagents, catalog #163-3003.) When using the ProteoMiner sequential elution kit, the elution reagent and rehydration reagent supplied with the ProteoMiner kit are not needed.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 15 13. Remove bottom cap, place column in collection tube labeled F4 and centrifuge at 1,000 x g for 30–60 sec to collect the elution. This elution contains your eluted proteins. Do not discard. 14. Repeat steps 12 and 13 two more times and collect both eluents in tube F4. 15. Store elutions at –20°C or proceed with downstream analysis. (See Section 11 for more information on preparing sample for analysis.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 16 Volume of sample available Volume of 20% bead slurry required Resulting settled bead volume (after swelling solution is removed) 200 µl 100 µl 20 µl 500 µl 250 µl 50 µl 1,000 µl 500 µl 100 µl This chart assumes a protein concentration of >50 mg/ml, which is typical for serum. If your sample concentration is lower, adjust volumes accordingly to assure 0.5 mg of protein per µl of beads.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 17 Section 11 Appendix Preparation for Various Downstream Applications Prior to any downstream analysis, you will need to quantitate the amount of protein in your sample. For this we recommend the Quick Start™ Bradford protein assay (Catalog #500-0201). If your downstream analysis technique is negatively impacted by low pH or salts, we recommend that you clean up your sample using Bio-Rad's ReadyPrep™ 2-D cleanup kit (catalog #163-2130).
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 18 Section 12 References Boschetti E et al., Reduction of dynamic protein concentration range of biological extracts for the discovery of low-abundance proteins by means of hexapeptide ligand library, Nature Protocols 3 (5), 883–890 (2008). Boschetti E et al., Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands, Electrophoresis 26, 3561–3571 (2005). Boschetti E et al.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page 19 ProteoMiner Kit Accessories Catalog# Description 163-3003 ProteoMiner Sequential Elution Reagents, 10 preps, includes reagents only (columns not included), to be used with 163-3006 or 163-3007 163-3012 ProteoMiner Dry Bulk Beads, 0.
10010636D:4110137B-4.0.GR.qxd 3/12/2009 7:51 AM Page COV1 This product is optimized for use in fractionating proteins and is covered by patents owned exclusively by Bio-Rad Laboratories and The American Red Cross. No license under these patents to use this product in ligand identification applications is conveyed expressly or by implication to the purchaser by the purchase of this product.
