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4110063A.qxd 1/29/2003 2:48 PM Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
4110063A.qxd 1/29/2003 2:48 PM Page 3 Table of Contents Section 1 Introduction……………………………………...1 Section 2 Kit Specifications…………………………......2–3 Section 3 Storage Conditions………………………..........3 Section 4 Instructions for Use………………….............4–6 Section 5 Appendix……………..……………..............7–11 Section 6 References…………..……………..................12 Section 7 Product Information………………….........
110063A.qxd 1/29/2003 2:48 PM Page 4 Section 1 Introduction The ReadyPrep reduction-alkylation kit offers an easy and effective way of improving spot resolution during 2-D electrophoresis. This general-purpose kit minimizes streaking and aberrant spot migration by reducing and irreversibly blocking the formation of inter- and intramolecular disulfide bonds prior to isoelectric focusing (IEF).
4110063A.qxd 1/29/2003 2:48 PM Page 5 Section 2 Kit Specifications The components provided in the ReadyPrep reductionalkylation kit are listed below. Each kit contains sufficient reagent to perform a reduction-alkylation reaction on 100 samples of 300 µl each containing up to 10 mg/ml of protein. Items Supplied With Kit 3 ampoules TBP (200 mM), 0.
4110063A.qxd 1/29/2003 2:48 PM Page 6 Items Required But Not Provided: • 1.5 ml microcentrifuge tubes • Microcentrifuge capable of spinning at 12,000–16,000 x g • Rehydration/sample buffer Section 3 Storage Conditions Store the unopened kit at room temperature. Once opened, follow the storage instructions provided on each component. Once an ampoule of TBP has been opened, transfer the TBP to one of the empty glass vials provided and store at -20°C.
4110063A.qxd 1/29/2003 2:48 PM Page 7 Section 4 Instructions for Use Per 500 µl sample volume: Note: This protocol can be scaled for smaller and larger protein sample volumes by adjusting the amounts of alkylation buffer, TBP, and iodoacetamide solution accordingly. 1. Prepare the iodoacetamide solution immediately before use by dissolving the contents of one vial in 0.6 ml of ultrapure water. Bio-Rad ReadyPrep proteomic grade water (catalog #163-2091) is recommended.
4110063A.qxd 1/29/2003 2:48 PM Page 8 For best results, the protein concentration of the sample should be determined. We recommend the Bio-Rad RC DC™ Protein Assay for protein quatitation because the assay is suitable for the widest variety of samples and works in the presence of detergents, reducing agents, and other agents that interfere with other protein assays. Note: The protein sample must be free of reducing agent (i.e., TBP, DTT, or TCEP) before the reduction-alkylation reaction is performed.
4110063A.qxd 5. 1/29/2003 2:48 PM Page 9 Add 15 µl of 0.5 M iodoacetamide for a final concentration of 15 mM. Mix well by vortexing. Incubate at room temperature for 1 hr. Note: Longer incubation times may cause spurious reactions between the iodoacetamide and the protein that could create artifacts in mass spectrometry analysis. 6. Add an additional 12.5 µl of TBP to quench any unreacted iodoacetamide. Mix well by vortexing. Incubate at room temperature for 15 min. 7.
4110063A.qxd 1/29/2003 2:48 PM Page 10 Section 5 Appendix 5.1 2-D Rehydration/Sample Buffer Volume The table that follows indicates appropriate volumes of 2-D rehydration/sample buffer needed to rehydrate IPG strips of specific lengths and the approximate amounts of protein required for detection using silver stain or Coomassie Blue G-250 stain. When loading the protein onto the IPG strips by cup loading, refer to the Cup Loading Tray instruction manual (4006216) for detailed instructions.
4110063A.qxd 1/29/2003 2:48 PM Page 11 5.2 Preparation of 2-D Rehydration/Sample Buffers The 2-D rehydration/sample buffer is not a component of this kit, but is necessary for performing 2-D electrophoresis. It is not provided in the kit because different protein samples can require different rehydration/sample buffers. For convenience, a selection of 2-D rehydration/sample buffer formulas are provided below. Do not add reducing agent (i.e.
4110063A.qxd 1/29/2003 2:48 PM Page 12 5.2.1. General-Purpose 2-D Rehydration/Sample Buffer (no reducing agent) (8 M urea, 2% CHAPS, 0.2% Bio-Lyte® 3/10 ampholyte, 0.002% bromophenol blue) Component Final Concentration Amount to Make 2 ml Urea (FW 60.06) CHAPS 100X Bio-Lyte 3/10 ampholyte** Bromophenol Blue 8M 2% (w/v) 0.2% (w/v) 0.96 g 0.04 g 20 µl 0.002% (w/v) 4 µl of a 1% (w/v) solution 1.
4110063A.qxd 1/29/2003 2:48 PM Page 13 Component Final Concentration Amount to Make 2 ml Urea (FW 60.06) Thiourea CHAPS* 100X Bio-Lyte 3/10 ampholyte*** Bromophenol Blue 7M 2M 4% (w/v) 0.2% (w/v) 0.84 g 0.304 g 0.08 g 20 µl 0.002% (w/v) 4 µl of a 1% (w/v) solution 1.1 ml Proteomic grade water *Other neutral or zwitterionic detergents can also be used at concentrations of 1% to 2% (w/v) to improve solubilization of membrane and hydrophobic proteins.
4110063A.qxd 1/29/2003 2:48 PM Page 14 complete 2-D rehydration/sample buffer, add 1.1 ml of PSB diluent to each 1 g of PSB powder. Note: Before weighing out the PSB powder, shake the bottle vigorously for 10–15 sec to break up any clumps and to ensure a uniform blend of the different components. Mix the solution until the powder is completely dissolved (the tube can be warmed to speed dissolution of the solids, but do not allow the temperature to exceed 30°C).
4110063A.qxd 1/29/2003 2:48 PM Page 15 Section 6 References Görg A et al., The current state of two-dimensional electrophoresis with immobilized pH gradients, Electrophoresis 21, 1037–1053 (2000) Hoving S et al., Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients, Proteomics 2, 127 (2002) Herbert B et al.
4110063A.qxd 1/29/2003 2:48 PM Page 16 Section 7 Product Information Catalog # Description Buffer Components 161-0460 CHAPS, 1 g 161-0731 Urea, 1 kg 161-0716 Tris, 500 g 161-0302 Sodium Dodecyl Sulfate (SDS), 1 kg 163-2094 100X Bio-Lyte 3/10 Ampholyte, 1 ml 163-2093 100X Bio-Lyte 7/10 Ampholyte, 1 ml 163-2091 ReadyPrep Proteomic Grade Water 163-2101 Tributylphosphine (TBP), 200 mM, 0.
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