Model 491 Prep Cell Instruction Manual For Technical Service call your local Bio-Rad office or in the U.S. call 1-800-424-6723.
Note To ensure the best performance from the Model 491 prep cell, become fully acquanted with these operating instructions before using the cell to transfer samples. Bio-Rad recommends that you first read these instructions carefully. Then assemble and disassemble the cell completely without transferring sample. After these preliminary steps, you should be ready to transfer a sample.
Table of Contents Section 1 1.1 1.2 1.3 1.4 1.5 General Information..................................................................................1 Introduction.................................................................................................1 Accessory Equipment.................................................................................1 Specifications.............................................................................................2 Chemical Compatability................
Section 9 9.1 9.2 9.3 9.4 9.5 A Guide to Preparative Native-PAGE.....................................................33 Introduction...............................................................................................33 How to Choose Native-PAGE Systems.....................................................33 Discontinuous Native-PAGE......................................................................36 Continuous Native-PAGE........................................................................
Section 1 General Information 1.1 Introduction The Model 491 prep cell* is designed to purify proteins or nucleic acids from complex mixtures by continuous-elution electrophoresis. Conventional gel electrophoresis buffer systems and media are used with the prep cell. During a run, samples are electrophoresed through a cylindrical gel. As molecules migrate through the gel matrix, they separate into ring shaped bands.
1.3 Specifications Construction Upper buffer chamber Lower buffer chamber Electrodes Lid Gel tube assembly Elution chamber base Elution frit Support frit Cooling core Elution tube Casting stand Shipping weight Overall size Voltage limit Current limit Power limit Cooling buffer flow rate Elution buffer flow rate Upper electrophoresis buffer volume Elution buffer chamber volume Lower electrophoresis buffer volume acrylic acrylic platinum, 0.
The buffer recirculation pump is also ground isolated as should be any pump used with this cell. During normal operation, the buffer in the lower buffer chamber is circulated through the cooling core and routed back into the system via the buffer recirculation pump. The buffer flowing through the tubing and the pump is electrically active. For this reason handle the tubing carefully while the power supply is on. Do not touch any exposed liquid when the power supply is on.
Section 2 Description of Major Components 2.1 Model 491 Prep Cell Components Lid & power cables Cooling buffer outlet Elution buffer outlet Upper buffer chamber Cooling core Elution buffer feedline Gel assembly tube Elution chamber cap Fluted gasket Elution frit Dialysis membrane Support frit O-ring Elution chamber base Lower buffer chamber Cooling buffer inlet Fig. 1. Exploded view of the Model 491 prep cell.
2.2 Lower and Upper Buffer Chamber The lower buffer chamber forms a stable base for the unit. It houses the anode and contains the lower electrophoresis buffer. The upper buffer chamber holds the upper electrophoresis buffer and the elution buffer, and houses the cathode. 3 4 6 5 8 7 1 2 Fig. 2. Upper and lower buffer chamber. Lower buffer chamber: Stopcock for lower electrophoresis buffer inlet (1), and anode (2).
2.4 Gel Tube Assembly The gel tube assembly holds both the gel and the cooling core. The elution chamber cap and the gasket mounted on the graduated gel column make up the upper part of the elution chamber. Two gel tube assemblies are provided with the Model 491 prep cell: 28 mm ID and 37 mm ID. See Section 4.4 for selecting the appropriate gel tube size for specific applications. 5 4 7 1 8 6 3 2 Fig. 4. Gel tube assembly. Cooling core collar (1), upper reservoir attachment, i.e.
2.6 Casting Stand Gels are cast with the gel tube assembly mounted directly on the casting stand. The casting stand ensures that gels have perfectly flat lower surfaces. Inserting a spatula in the gel-release slot facilitates the removal of the gel from the casting stand after polymerization by allowing air to enter beneath the gasket and the gel. 2 3 1 4 Fig. 6. Casting stand. Leveling feet (1), center pin (2), leveling bubble (3), and gel release slot (4).
Section 3 Assembly and Operation 3.1 Casting the Preparative Gel 1. Place the gel tube assembly on the casting stand, aligning the four screws on the acrylic plate with the holes in the casting stand. Secure the gel tube assembly with the four screws; hand tightening is sufficient. Level the casting stand with the aid of the leveling bubble using the leveling legs. 2.
3. It is advisable to cool the gel during polymerization. Cooling prevents excess heat accumulation in the interior of the reaction mixture and aids in the formation of uniform gels. To cool, pump room temperature water (or buffer) from an external source through the cooling core. Ensure that cooling is in progress prior to casting the gel. Fig. 7. Diagram of cooling path during polymerization. Cooling of gels during polymerization is recommended.
3.2 Preparing the Frits and Dialysis Membrane Soak the elution manifold support frit, elution frit, and dialysis membrane in buffer. The frits must be completely wetted prior to use. To ensure removal of entrapped air in the pores of the frits, place the container in which the frits are soaking in a vacuum chamber for approximately 10 minutes. Alternatively, the frits can be soaked in buffer overnight to completely wet them. To maintain the wetting of the frits, store them in buffer.
2. Decant the stacking gel overlay, rinse the surface of the stacking gel with water, and loosen the four screws holding the column to the casting stand. Carefully remove the gel tube assembly from the casting stand. Insert a spatula into the gel release slot and use it to gently pry the gel tube assembly off the casting stand. Inspect the lower surface of the gel to make sure it is smooth. Trapped air bubbles may cause a pitted gel surface which will result in uneven elution of proteins from the gel.
4. To simplify sample loading, the sample loading guide should be inserted into the space between the cooling core and the gel tube at this time. 3.4 Assembly of Upper and Lower Buffer Chambers 1. Carefully place the upper buffer reservoir on the gel tube assembly and seat it firmly. Make sure the small O-ring is properly seated in the threaded connector. The following connections should be made before the black ring nut is tightened.
3. Fill the upper electrophoresis buffer reservoir (300–600 ml) and the elution buffer reservoir (750 ml) and check that all the lines are properly connected. If leaking is observed, check connections and reconnect where necessary. 4. Thoroughly degas 1,500 ml electrophoresis/elution buffer. With the stopcock of the lower buffer tank closed, fill the lower buffer reservoir with buffer to cover at least the height of gel.
3.5 Purge the Elution Chamber of Air Bubbles The elution chamber must be purged of air prior to running the cell. To do this, attach a 50 ml syringe to the elution buffer outlet tubing at the top of the cooling core. Gently pull elution buffer through the elution chamber into the 50 ml syringe. To remove air bubbles trapped in the channels of the gray elution gasket, gently push buffer (not air) back into the elution chamber with a full syringe.
3.6 Cooling the Gel First, determine which setting on the buffer recirculation pump will provide a flow rate of ~100 ml/min. Next, using the tubing provided with the Model 491 prep cell, connect the outlet of the buffer pump to the inlet (stopcock) of the lower buffer chamber. Then connect the tubing exiting from the top of the cooling core to the inlet of the buffer pump. Open the valve and set the pump speed at the maximal setting for a few minutes to purge air from the lines.
3.7 Loading the Sample Carefully load the sample on the surface of the gel through the sample loading guide with the sample application syringe. Layer the sample under the electrophoresis buffer above the gel. Make sure the stacking gel is not punctured with the PTFE tubing. Once the sample is loaded, place the lid on the cell and attach the cables to the power supply. Set the power supply to the appropriate setting and begin electrophoresis. 3.
Section 4 Optimizing Running Conditions for Preparative SDS-PAGE The Model 491 prep cell is designed for separating a single component from its nearest contaminant. The conditions required to achieve optimum resolution may be different than those of analytical electrophoresis.
4.2 Optimization Procedures A. Simplified Optimization Procedure Optimum %T When the difference in the molecular weights of the protein of interest and its nearest contaminant is 10% or greater, select the monomer concentration (optimum %T) which corresponds to the size of the protein of interest from the plot below. In most cases the %T so obtained will provide adequate resolution for the purified protein. For gel length and gel tube size determination refer to Sections 4.3 and 4.4.
Table 1. Recommended monomer concentrations. Size Range %T Range 15–30 kD 6–10% 30–50 kD 9–12% 50–70 kD 7–10% 70–100 kD 5–9% 100-200 kD 4–8% The procedure goes as follows: 1. Cast 3–4 polyacrylamide mini-slab gels in the range suggested in Table 1 (and Figure 10). 2. Load a sufficient amount of protein for detection by silver staining (~100 ng/lane). Load at least one lane with 10 µl prestained high or low molecular weight standards and one lane with SDS-PAGE silver stain standards (optional).
The larger gel surface of the 37 mm tube also allows for greater sample loads than does the 28 mm tube. Tighter bands can be maintained by distributing the protein over a larger area. Refer to Table 2 to determine the correct tube size and gel length for your purification scheme. The following guidelines are established for optimizing resolution and recovery of the protein of interest with the least amount of dilution.
4.5 Running Conditions We recommend running SDS-PAGE gels in the Model 491 prep cell using 12 W constant power. Values for voltage and current should correspond to the settings listed in Table 4. Different running conditions apply for native-PAGE (Section 9). Table 4. Running conditions for preparative SDS-PAGE. 12 W Constant Power Small gel tube (28 mm ID) 40–50 mA/240–300 V Large gel tube (37 mm ID) 50–60 mA/200–400 V 4.
4.7 Examples of the SDS-PAGE Optimization Procedure The following two examples demonstrate optimization protocols for separating two closely spaced proteins on the Model 491 prep cell. In each example, a series of mini-slab gels were run to determine the best acrylamide concentration to use in the preparative gel. Example 1: Purification of the Subunits of Phycocyanin Phycocyanin, purified by ion exchange chromatography, contains two naturally colored blue protein subunits of ~18.
To confirm the procedure, the Model 491 prep cell was run using the same %T range as the analytical gels. Preparative run conditions are shown in Table 6. Table 6. Preparative run conditions. Gel Composition 12%–17% T/2.67% bis Gel Height 5.5 cm Gel Size 25 mm ID Sample Load 1 mg total protein Running Conditions 40 mA constant current (~250–350 V) Running Time ~5 hr For each run the resolution (R = peak separation/average peak width) was determined.
A kD 18.5 kd 21 kD kd Ionic contaminants 23 kD kd 10 20 30 1 hr 2 hr 3 hr 4 hr 5 hr B kD 23 21 18.5 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Fig. 13. Elution profile and SDS-PAGE analysis. A, Elution profile. Chromatographically purified phycocyanin was separated into three subunits (18.5 kD, 21 kD, and 23 kD) by preparative SDS-PAGE in the Model 491 prep cell.
Distance protein bands, Distance between between protein bands (mm ) mm Keyhole limpet hemocyanin: separation ofof 96kd and 98kd Keyhole limpet hemocyanin: separation 96 kD and 98 proteins kD proteins 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 4 5 6 7 %T 8 9 10 11 Fig. 14. Determination of optimal %T for 96 kD and 98 kD keyhole limpet hemocyanin proteins. The breakpoint of the curve is considered the monomer concentration for optimal separation. In this case a 7%T/2.
Section 5 Disassembling and Cleaning 1. Turn off the power supply, disconnect the cables from the power supply, and remove the lid. Turn off both the elution and cooling pumps. 2. Disconnect the elution buffer tubing from the cooling core. Close the valve of the cooling buffer outlet on the lower reservoir and disconnect the cooling pump tubing from the lower reservoir and the cooling core. 3.
Section 6 Troubleshooting Guide Problem Cause Solution 1. Sample requires a long time to enter the gel. a. High salt concentration in the sample. a. Remove salts by dialysis, desalting column, etc. 2. Poor resolution. a. Sample overloaded. a. Decrease sample load. b. Incorrect %T. b. Refer to section 4. c. Incorrect gel length or gel tube size. c. Check Table 2. d. Inadequate cooling. d. Cooling flow rate should be 100 ml/min. a. Proteins too dilute and UV detection not sufficient.
Section 7 Preparation of Electrophoresis Buffers and Acrylamide Stock Solutions for SDS-PAGE The Model 491 prep cell has three buffer reservoirs: one for the lower electrophoresis buffer (~3 liters), one for upper electrophoresis buffer (~600 milliliters) and one for the elution buffer (~900 milliliters). For SDSPAGE all three reservoirs contain the same buffer (Laemmli buffer system). It is recommended that 6.0 liters of buffer be prepared for each preparative run.
7.4 10x Electrode (Running) Buffer, pH 8.3 (Makes 1 L) Tris base Glycine SDS 30.3 g 144.0 g 10.0 g Dissolve and adjust to 1,000 ml with deionized water. DO NOT adjust pH with acid or base. To make 6 L of 1x electrophoresis buffer (0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3) for the prep cell, dilute 600 ml of 10x stock with 5,400 ml deionized water. 7.5 30% Acrylamide Stock Solution Acrylamide/bis (N,N'-bis-methylene-acrylamide) (30%T/2.67%C) Acrylamide Bis 146.0 g 4.
Section 8 SDS-PAGE Gel Preparation 8.1 Gel Recipes Table 8 can be used to prepare both analytical and preparative Tris-HCl acrylamide gels. Sections 8.2–8.3 provide detailed formulas for calculating specific gel percentages and volumes. Acrylamide/bis stock solution of 30% (37.5:1) are used. The amounts listed for the components in Table 8 are based on a total volume of 10 ml. Determine the total volume needed and multiply each component by the appropriate number.
8.2 Analytical Separating Gels Table 10. Calculating %T (0.375 M Tris, pH 8.8). Separating Monomer Concentration = %T = Acrylamide/bis (30% T/2.67%C Stock) c ml Deionized water d ml 1.5 M Tris HCI, pH 8.8 2.
8.4 Preparative SDS-PAGE Separating and Stacking Gels (The following calculations are based on the preparation of a full length separating gel of ~11 cm with a ~2.5 cm stacking gel in the 28 mm ID gel tube) Table 12. Calculating %T. Monomer Concentration Acrylamide/bis (30% T/2.67%C Separating Gel %x = %T Stacking Gel 4% T c ml 1.3 ml d ml 6.2 ml stock solution) Deionized water 0.5 M Tris HCI, pH 8.8 10.0 ml – 1.5 M Tris HCI, pH 6.8 – 2.5 ml 100 µl 50.
Section 9 A Guide to Preparative Native-PAGE 9.1 Introduction Conventional gel electrophoresis buffer systems and media are used with the Model 491 prep cell to separate individual components from their nearest contaminants. This guide describes a method for selecting the best nondenaturing PAGE system to isolate a particular protein with the Model 491 prep cell. Native-PAGE Theory Preparative native-PAGE is a technique for high yield purification of biologically active molecules.
Continuous Buffer Systems The pH attained in the resolving gel of the Ornstein-Davis system approaches pH 9.5, which may be outside the range of stability for some proteins. Alternative discontinuous buffer systems derived for preparative work can be found in an article by Chrambach and Jovin (Chrambach and Jovin 1984). The electrophoresis buffers described in this article span the pH range from 3–10.
Determine the pI of the protein under investigation. Isoelectric focusing is recommended for this purpose. Determine the pH range where the protein retains its biological activity. Is the protein stable at pH 9.5? YES Is the pI of the protein below pH 8.5? Ornstein-Davis buffer system (pH 9.5) should not be used because: 1. The protein of interest will denature due to high pH. 2. Or, the protein of interest will carry so little net charge that it will not migrate through the gel. 3.
9.3 Discontinuous Native-PAGE Acrylamide Concentration – Gel Pore Size By convention, polyacrylamide gels are characterized by the figures (%T/%C), where %T is the weight percentage of total monomer including crosslinker (in g/100 ml) and %C is the proportion of crosslinker as a percentage of total monomer. For both the analytical gels and the preparative gels use 2.67% N, N’-methylenebis acrylamide crosslinker (premixed acrylamide:bis in the ratio 37.5:1 can also be used).
Preparing Ornstein-Davis Native Electrophoresis Buffer Solutions A. Resolving (Separating) Gel Buffer (1.5 M Tris-HCl pH 8.8) Dissolve 27.23 grams Tris base in approximately 80 ml deionized water. Adjust to pH 8.8 with 6 N HCl. Make to 150 ml with deionized water and store at 4˚C. B. Stacking Gel Buffer (0.5 M Tris-HCl, pH 6.8) Dissolve 6 grams Tris base in approximately 60 ml deionized water. Adjust to pH 6.8 with 6 N HCl. Make to 100 ml with deionized water and store at 4 ˚C. C. Sample Buffer (0.
Prepare Ornstein-Davis Acrylamide Gels Use the following table to prepare acrylamide gels (both analytical and preparative). The amounts listed for the components in the table below are based on a total volume of 10 ml. Determine the total volume needed and multiply each component by the appropriate number. Table 13. Reference table for the preparation of acrylamide gels (10 ml volume). %T Deionized H2O, ml Gel Buffer*, ml Bis-acrylamide solution 30% stock (37.5:1), ml 4 6.15 2.50 1.33 5 5.
D. The migration rates of proteins run in Ornstein-Davis gels at 12 W constant power will approximate those shown in Table 15. Table 15. Approximate migration rates of proteins run in Ornstein-Davis gels at 12 W constant power. Rf Migration Rate 1.0 30 mn/cm gel 0.8 40 mn/cm gel 0.6 50 mn/cm gel 0.45 60 mn/cm gel Rf values for specific proteins are obtained from the mini-gels that were run to optimize conditions for the Model 491 prep cell.
Continuous Electrophoresis Buffers McLellan describes various continuous buffer systems from pH 3.8 to pH 10.2.3 (McLellan 1982). Use the table below to prepare 1 liter of 5x continuous nondenaturing PAGE buffer. DO NOT adjust pH with acid or base. If the final pH is outside the the listed range discard the buffer and remake. Table 16. Nondenaturing PAGE buffer preparation. Buffer pH ± 0.1 Basic Component and MW 5x Solution g/L or ml/L Acidic Component and MW 5x Solutions ml/L or g/L 3.
Prepare Resolving Gels As protein mobilities are best modified by pH, continuous nondenaturing PAGE systems use relatively large pore size gels. Generally 4–6 cm long gels are sufficient for optimum resolution in the prep cell. For 10 ml acrylamide monomer solution Table 18. Acrylamide monomer solution per 10ml of gel volume. %T Deionized H2O, ml Continuous Buffer*, ml Acrylamide/bis solution 30% stock (37.5:1), ml 4 6.65 2.00 1.33 5 6.30 2.00 1.67 6 5.85 2.00 2.
9.5 References Allen RC et al. (1984). Gel Electrophoresis and Isoelectric Focusing of Proteins: Selected Techniques. (New York: de Gruyter). Andrews AT (1986). Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications (Oxford: Clarendon Press). Chrambach A and Jovin T (1983). Selected buffer systems for moving boundary electrophoresis on gels at various pH values, presented in a simplified manner. Electrophoresis 4, 190–204. Hames BD (1990).
Section 10 Ordering Information Catalog Number Product Description 170-2925 Model 491 Prep Cell, without buffer recirculation pump 170-2926 Model 491 Prep Cell, with buffer recirculation pump (100/120VAC) 170-2927 Model 491 Prep Cell, with buffer recirculation pump (220/240VAC) 170-2929 Buffer Recirculation Pump, 120/100 VAC 170-2930 Buffer Recirculation Pump, 220/240 VAC Replacement Accessories 170-2932 Small Gel Tube Assembly, 28 mm ID 170-2933 Large Gel Tube Assembly, 37 mm ID 170-2934 Cooling Fi
Electrophoresis Chemicals Catalog Quantity/ Number Product Description Package Premixed Electrophoresis Buffers 161-0732 161-0755 10x Tris/Glycine/SDS Buffer 10x Tris/Glycine/SDS Buffer 6 x 1L 1L Premixed Acrylamide/bis 161-0122 161-0125 37.5:1 mixture, (2.67% C) 37.5:1 mixture, (2.
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