4006167b.qxd 08/28/2000 6:43 PM Page Cvr1 ReadyPrep™ Sequential Extraction Kit Instruction Manual Catalog # 163-2100 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
4006167b.qxd 08/28/2000 6:43 PM Page i Table of Contents Section 1. Introduction...........................................1 Section 2. Kit Components.....................................2 Section 3. Storage ..................................................3 Section 4. Reagent Preparation..............................3 Section 5. Instructions for Use...............................5 Section 6. References ..........................................15 Section 7. Product Information ...........................
4006167b.qxd 08/28/2000 6:43 PM Page 1 Section 1. Introduction The ReadyPrep sequential extraction kit provides the reagents necessary to extract proteins of differing solubility from cell lysates in a form suitable for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). It is based on the work of Molloy et al. [1] and Herbert et al. [2]. Each of the three solutions in this kit solubilizes a different set of proteins. Reagent 1 extracts only the most soluble proteins, such as cytosolic proteins.
006167b.qxd 08/28/2000 6:43 PM Page 2 Sequential extraction can be carried out in a single microcentrifuge tube, minimizing protein loss. The kit is particularly well suited for use with 2-D PAGE systems employing immobilized pH gradients for the first-dimension IEF. Section 2. Kit Components Reagent 1. One vial. Lyophilized. Each vial of rehydrated Reagent 1 contains 50 ml of 40 mM Tris base. Reagent 2. Three vials. Lyophilized.
4006167b.qxd 08/28/2000 6:43 PM Page 3 Section 3. Storage Store the lyophilized contents of the kit, Reagents 1, 2, and 3, unopened at room temperature. Store the sealed ampoule of reducing agent TBP unopened at room temperature. Section 4. Reagent Preparation Reagent 1: Add 50 ml of deionized water to the vial of Reagent 1. Swirl the vial gently at intervals until the contents are completely dissolved. Reconstituted Reagent 1 consists of 50 ml of 40 mM Tris base per vial.
4006167b.qxd 08/28/2000 6:43 PM Page 4 necessary to hold the vial in a bath of warm (tap) water for more efficient warming. Rehydrated Reagent 3 consists of 10 ml of 5 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (w/v) SB 3-10, 40 mM Tris, and 0.2% (w/v) Bio-Lyte 3/10 per vial. Storage of rehydrated solution 1, 2, 3. Following rehydration, aliquot the solutions as convenient and store the aliquots at -20 °C or lower. A convenient aliquot size is 1 ml, each placed into a microcentrifuge tube for storage.
4006167b.qxd 08/28/2000 6:43 PM Page 5 Section 5. Instructions for Use General Guidelines. The goal of the first step is to lyse the cells of interest directly in Reagent 1 using a physical lysis procedure. The method of lysis will vary greatly depending on cell type. Follow standard procedures for cell growth and tissue harvesting and standard methods for physical cell lysis. For example, bacterial cultures can be harvested by centrifugation then washed in Reagent 1 to remove excess medium.
4006167b.qxd 08/28/2000 6:43 PM Page 6 suspension of 0.5 g (wet weight — equivalent to about 100 mg dry weight) of Escherichia coli in 2 ml of Reagent 1 results in effective lysis and fractionation. Lysis conditions are very important to the success of a sequential extraction. Proper conditions for thorough lysis should be determined empirically. For example, overly aggressive sonication can denature some proteins, while insufficient sonication can leave some of the cells intact.
4006167b.qxd 08/28/2000 6:43 PM Page 7 with the Bio-Rad protein assay (Bradford), catalog # 500-0001, or the modified assay shown below. 5 Wash the insoluble pellet from Step 3 twice with the same volume of Reagent 1 used in Step 1. It is valuable to determine the protein concentrations in the washes. Discard the washes. 6 Store the supernatant frozen until it is used in 2-D PAGE.
4006167b.qxd 08/28/2000 6:43 PM Page 8 3 Vortex the mixture for 5 min. For some samples, it may be necessary to also sonicate the suspension or to aspirate it through a fine-gauge needle to solubilize the protein. 4 Centrifuge the mixture to give a firm pellet and a clear supernatant. 5 Recover the supernatant and determine its protein concentration. The modified Bio-Rad protein assay procedure shown below is recommended for determining protein concentrations in extraction solution 2.
4006167b.qxd 08/28/2000 6:43 PM Page 9 2 Use extraction solution 3 to solubilize proteins in the pellet from Extraction 2. Use approximately the same volume of extraction solution 3 as was used of extraction solution 2. Empirically determine the best volume for the third extraction. 3 Vortex and centrifuge as described above. 4 Recover the supernatant and determine its protein content.
4006167b.qxd 08/28/2000 6:43 PM Page 10 An example of the sequential extraction of an E. coli preparation is shown in the figure.
4006167b.qxd 08/28/2000 6:43 PM Page 11 A B C D Two-dimensional gel analysis of extracts from E. coli. E. coli W3110 was collected by centrifugation. The cell pellet was suspended in Reagent 1 and the cells were lysed by sonication. One portion of the sonicated cell suspension, containing 200 µg of protein, was diluted in extraction solution 3. The proteins soluble in solution 3 were separated by 2-D PAGE as a “whole cell extract” (A).
4006167b.qxd 08/28/2000 6:43 PM Page 12 Modified Bio-Rad Protein Assay This modification to the standard Bio-Rad protein assay procedure (catalog # 500-0001) is recommended to determine the protein content of Extracts 2 and 3 (modified from [10] and [11]). It is not necessary with Extracts 1, but it can be used with Extracts 1, the content of the samples, not the procedures. The high concentrations of detergents and chaotropes in Reagents 2 and 3 interfere with many other types of assays.
4006167b.qxd 08/28/2000 6:43 PM Page 13 4 Prepare a standard curve covering the range of 2–280 µg. • Dilute the BgG standard with extraction solution in a twofold (or other) dilution series from 14 to 0.1 mg/ml. TBP can be omitted from the standard-curve dilutions. The standard curves generated with gamma globulin in the three extraction solutions are similar.
4006167b.qxd 08/28/2000 6:43 PM Page 14 Very high-concentration extracts, with protein levels above the upper limit of the standard curve, require that lesser volumes of them be assayed. In such cases, increase the amount of 0.12 N HCl (nominal) used so that the total volume is 100 µl. • Add 3.5 ml of diluted dye reagent to each tube. Vortex gently. • Measure the absorbances at 595 nm (A 595 ) after 5 minutes.
4006167b.qxd 08/28/2000 6:43 PM Page 15 Section 6. References 1 2 3 4 5 6 7 8 9 10 11 Molloy, MP, et al. Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis. 1998 May;19(5):837-44. Herbert, BR, et al. Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis, 1998 May;19(5):845-51. Rabilloud, T, et al.
4006167b.qxd 08/28/2000 6:43 PM Page 16 Section 7. Product Information Catalog # Product Description 163-2100 ReadyPrep Sequential Extraction Kit 163-2101 Reducing Agent TBP, 200 mM, 0.
4006167b.qxd 08/28/2000 6:43 PM Page Cvr2 Bio-Rad Laboratories 2000 Alfred Nobel Dr.
