Mini-PROTEAN® Tetra Cell Instruction Manual Catalog Numbers 165-8000 165-8001 165-8002 165-8003 165-8004 165-8005 165-8006 165-8007
Table of Contents Section 1 General Information 1.1 Introduction 1.2 Components 1.3 Specifications 1.4 Safety 1 1 1 4 5 Section 2 Setup and Basic Operation 2.1 Gel Cassette Preparation 2.2 Electophoresis Module Assembly and Sample Loading 6 6 9 Section 3 Separation Theory and Optimization 3.1 Introduction 3.2 SDS-PAGE (Laemmli) Buffer System 3.3 Native PAGE 15 15 16 17 Section 4 Reagent Preparation and Stock Solutions 4.1 Volumes Required per Gel 4.2 SDS-PAGE (Laemmli) Buffer System 4.
Section 1 General Information 1.1 Introduction The Mini-PROTEAN® Tetra cell runs both handcast gels and Ready Gel® precast gels interchangeably. The Mini-PROTEAN Tetra system includes a casting stand and glass plates with permanently bonded gel spacers that simplify handcasting and eliminate leaking during casting. The cell can run one or four gels, and the mini tank is compatible with other Bio-Rad electrode modules for tank blotting, 2-D electrophoresis, and electroelution. 1.
Electrode Assembly The electrode assembly holds the gel sandwich. It houses the sealing gasket, the upper and lower electrodes, and the connecting banana plugs. The anode (lower electrode) banana plug is identified with a red marker and the cathode (upper electrode) banana plug with a black marker. Companion Assembly The companion assembly allows you to run gels 3 and 4. It holds the gel sandwich and houses the sealing gasket.
Lid Banana plug jacks Notch on U-shaped gasket Gel cassette Electrode assembly Mini tank Fig. 1. Assembling the Mini-PROTEAN Tetra Cell. Fig. 2.
1.3 Specifications Casting Stand* Polycarbonate Pin, retaining ring, and spring Casting Frames* Stainless steel Polysulfone Gray gaskets Thermoplastic rubber (gray) Electrode Assembly Glass-filled polybutylene terephthalate Electrodes Platinum wire, 0.
Chemical Compatibility Mini-PROTEAN Tetra cell components are not compatible with acetone or ethanol. Use of organic solvents voids all warranties. Call 1-800-4-BIORAD (US) or your local Bio-Rad representative for technical information regarding chemical compatibility of the Mini-PROTEAN Tetra cell with various laboratory reagents. The Mini-PROTEAN are not compatible with repeated exposure to 100% TEMED.
Section 2 Setup and Basic Operation 2.1 Gel Cassette Preparation Handcast Gels 1. Glass Cassette and Casting Stand Assembly Note: All glass plates should be clean and dry. a. Place the casting frame upright with the pressure cams in the open position and facing forward on a flat surface. b. Select a spacer plate of the desired gel thickness and place a short plate on top of it (see Figure 3a). c. Orient the spacer plate so that the labeling is up.
f. Repeat steps a–e for additional gels. 3a 3b 3c 3d Fig. 3. Assembling the Mini-PROTEAN casting stand and frame. 2.0 Gel Casting a. Discontinuous Polyacrylamide Gels i. Place a comb completely into the assembled gel cassette. Mark the glass plate 1 cm below the comb teeth. This is the level to which the resolving gel is poured. Remove the comb. ii. Prepare the resolving gel monomer solution by combining all reagents except APS and TEMED. (Refer to section 4 for gel formulations.
iv. Immediately overlay the monomer solution with water or t-amyl alcohol. Note: If water is used, add it slowly and evenly to prevent mixing. Do not overlay with butanol or isobutanol. v. Allow the gel to polymerize for 45 min to 1 hr. Rinse the gel surface completely with distilled water. Do not leave the alcohol overlay on the gel for more than 1 hr because it will dehydrate the top of the gel. Note: At this point the resolving gel can be stored at room temperature overnight.
Ready Gel® Precast Gels 1. Ready Gel Cassette Preparation Note: The Mini-PROTEAN Tetra cell is guaranteed for use with Bio-Rad’s Ready Gel and Mini-PROTEAN® precast gels. For more information, go to www.bio-rad.com/mpgels. a. Remove the Ready Gel from the storage pouch. b. Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer. c. Cut along the dotted line at the bottom of the Ready Gel cassette with a razor blade. d.
a. Set the clamping frame to the open position on a clean flat surface (see Figure 4a). b. Place the first gel sandwich or gel cassette (with the short plate facing inward) onto the gel supports; gel supports are molded into the bottom of the clamping frame assembly; there are two supports in each side of the assembly. Note that the gel will now rest at a 30° angle, tilting away from the center of the clamping frame.
Important Note: Do not attempt to lock the green arms of the clamping frame, without first ensuring that the gel cassettes are perfectly aligned and stabilized against the notches on the green gaskets of the module. To prevent the gels from shifting during the locking step, firmly and evenly grip them in place against the core of the module with one hand. Caution: When running 1 or 2 gels only, do not place the companion running Module in the tank.
2. Sample Loading a. Fill the assembly (upper chamber) with buffer to just under the edge of the outer gel plate. b. Load samples into each of the assemblies while they are sitting on a flat surface, outside of the tank. c. Load the samples into the wells with a Hamilton syringe or a pipet using gel loading tips. d. If using Bio-Rad’s patented sample loading guide, place it between the two gels in the electrode assembly. Sample loading guides are available for 9, 10, 12, and 15-well formats. e.
b. If running 2 gels only, you will be using just the electrode assembly, so place this assembly in the back position of the cell, making sure that the red (+) electrode jack matches the red marking on the top right inside edge of the tank. c. If running 4 gels, place the electrode assembly (banana plugs) in the back position (as detailed in 2.2.3b.) and the companion running module (no banana plugs) in the front position.
6. Gel Removal a. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads. b. Remove the tank lid and carefully lift out the electrode assemblies. Pour off and discard the running buffer. Note: Always pour off the buffer before opening the arms of the assembly, to avoid spilling the buffer. c. Open the arms of the assembly and remove the gel cassettes. d. Remove the gels from the gel cassette by gently separating the two plates of the gel cassette.
Section 3 Separation Theory and Optimization 3.1 Introduction Polyacrylamide gel electrophoresis separates molecules in complex mixtures according to size and charge. During electrophoresis there is an intricate interaction of samples, gel matrix buffers, and electric current resulting in separate bands of individual molecules. Hence the variables that must be considered in electrophoresis are gel pore size, gel buffer systems, and the properties of the molecule of interest.
The total monomer concentration for optimal separation is referred to as optimal %T. Optimal %T will vary depending on the molecular weight of the molecule of interest. Empirically the pore size providing optimum resolution for proteins is that which results in a relative mobility (Rf) value between 0.55–0.6. Rf values for specific proteins are calculated as follows.
to their molecular weights. Proteins are separated according to their molecular weights, making this system extremely useful for calculating molecular weights. 3.3 Native PAGE Native PAGE is a technique for separating biologically active proteins. In contrast to SDS-PAGE, the mobilities of proteins in a native PAGE system depend on both size and charge. There is no single electrophoresis buffer system that will optimally purify all native proteins.
Note: 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, causing denaturation. Additionally, the pI of the protein of interest may be too close to or above the Ornstein-Davis buffer pH (9.5), which may result in a very low net charge or a positive net charge that may significantly reduce or even prohibit migration to the anode.
Section 4 Reagent Preparation and Stock Solutions 4.1 Volumes Required Per Gel The volumes listed are required to completely fill a gel cassette. Amounts may be adjusted depending on the application (with or without comb, with or without stacking gel, etc.). Gel Thickness (mm) Volume (ml) 0.5 2.8 0.75 4.2 1.0 5.6 1.5 8.4 Note: 10 ml of monomer solution is sufficient for two stacking gels of any thickness. 4.2 SDS-PAGE (Laemmli) Buffer System Stock Solutions and Buffers 1. Acrylamide/Bis (30%T, 2.
Adjust to pH 8.8 with 6 N HCl. Bring total volume to 150 ml with deionized water and store at 4°C. Alternatively, 1.5 M Tris-HCl, pH 8.8 (1 L) premixed buffer can be used (Bio-Rad catalog #161-0798). 4. 0.5 M Tris-HCl, pH 6.8 6g 60 ml Tris base deionized water Adjust to pH 6.8 with 6 N HCl. Bring total volume to 100 ml with deionized water and store at 4°C. Alternatively, 0.5 M Tris-HCl, pH 6.8 (1 L) premixed buffer can be used (Bio-Rad catalog #161-0799). 5. Sample buffer (SDS reducing buffer) 3.55 ml 1.
Gel Formulations (10 ml) 1. Prepare the monomer solution by mixing all reagents except the TEMED and 10% APS. Degas the mixture for 15 min. 30 % Degassed Percent DDI H2O Acrylamide/Bis Gel buffer 10% w/v SDS gel (ml) (ml) (ml) 4% 6.1 1.3 2.5 (ml) 0.1 5% 5.7 1.7 2.5 0.1 6% 5.4 2.0 2.5 0.1 7% 5.1 2.3 2.5 0.1 8% 4.7 2.7 2.5 0.1 9% 4.4 3.0 2.5 0.1 10% 4.1 3.3 2.5 0.1 11% 3.7 3.7 2.5 0.1 12% 3.4 4.0 2.5 0.1 13% 3.1 4.3 2.5 0.1 14% 2.7 4.7 2.5 0.
4.3 Discontinuous Native PAGE (Ornstein-Davis) Stock Solutions and Buffers 1. Acrylamide/Bis (30%T, 2.67%C) 87.6 g acrylamide (29.2 g/100 ml) 2.4 g N’N’-bis-methylene-acrylamide (0.8 g/100 ml) Make to 300 ml with deionized water. Filter and store at 4°C in the dark (30 days maximum). Or, use: Preweighed acrylamide/bis, 37.5:1 mixture (Bio-Rad catalog #161-0125, 150 g) 30% acrylamide/bis solution, 37.5:1 mixture (Bio-Rad catalog #161-0158, 500 ml) (Bio-Rad catalog #161-0159, 2 x 500 ml) 2. 1.
5. 10x electrode (running) buffer, pH 8.3 30.3 g Tris base (15 g/L) 144.1 g glycine (72 g/L) Bring total volume up to 1,000 ml with deionized water. Do not adjust pH. Alternatively, electrophoresis running buffer 10x Tris/ glycine, 1 L (Bio-Rad catalog #161-0734) can be used. Usage: Dilute 100 ml of 10x stock with 900 ml deionized water for each electrophoresis run. Gel Formulations (10 ml) 1. Prepare the monomer solution by mixing all reagents except the TEMED and 10% APS. Degas the mixture for 15 min.
4.4 Continuous Native PAGE Stock Solutions and Buffers 1. Acrylamide/Bis (30%T, 2.67%C) 87.6 g acrylamide (29.2 g/100 ml) 2.4 g N’N’-bis-methylene-acrylamide (0.8 g/100 ml) Make to 300 ml with deionized water. Filter and store at 4°C in the dark (30 days maximum) Or use: Preweighed acrylamide/bis, 37.5:1 mixture (Bio-Rad catalog #161-0125, 150 g) 30% acrylamide/bis solution, 37.5:1 mixture (Bio-Rad catalog #161-0158, 500 ml) (Bio-Rad catalog #161-0159, 2 x 500 ml) 2. Sample Buffer 1.0 ml 3.0 ml 0.2 ml 5.
Basic pH Acidic Component 5x Solution Component 5x Solution 3.8 ß-Alanine 13.36 g/L Lactic acid 7.45 ml/L (MW 89.09) 4.4 ß-Alanine 85% solution 35.64 g/L (MW 89.09) 4.8 GABA Histidine 41.24 g/L Imidazole 23.28 g/L Tris 19.94 g/L Tris 14.64 g/L Tris 19.38 g/L Tris 30.28 g/L Ammonia 31.4 g/L HEPES 41.7 g/L EPPS 37.85 g/L Boric acid 7.73 g/L 9MW 61.83) 36.34 g/L (MW 121.14) 1.0 MOPS (MW 252.2) (MW 121.14) 9.4 29.5 g/L (MW 238.33) (MW 121.14) 8.7 MES (MW 209.
Dilute 200 ml of 5x buffer with 800 ml deionized water to prepare 1x electrophoresis buffer. The final concentrations of buffer components will be. pH Basic component Acidic component 3.8 30 mM ß-Alanine 20 mM Lactic acid 4.4 80 mM ß-Alanine 40 mM Acetic acid 4.8 80 mM GABA 20 mM Acetic Acid 6.1 30 mM Histidine 30 mM MES 6.6 25 mM Histidine 30 mM MOPS 35 mM HEPES 7.4 43 mM Histidine 8.1 32 mM Tris 30 mM EPPS 8.7 50 mM Tris 25 mM Boric acid 9.4 60 mM Tris 40 mM CHAPS 10.
Section 5 References Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature 227, 680-685. Ornstein L (1964). Disc electrophoresis. I. Background and theory. Ann N Y Acad Sci 121, 321-349. Chrambach (1983). A and Jovin, T M, Electrophoresis, 4, 190–204 McLellan T (1982). Electrophoresis buffers for polyacrylamide gels at various ph. Anal Biochem 126, 94-99.
Section 7 Troubleshooting Guide Problem Cause Solution Smile effect – band pattern curves upward at both sides of the gel Center of the gel running hotter than either end Buffer not mixed well or buffer in upper chamber too concentrated.
Troubleshooting Guide (cont.
Troubleshooting Guide (cont.) Problem Cause Solution Poor end well formation Incorrect catalyst formation Prepare fresh catalyst solution, or increase the catalyst concentration of the stacking gel to 0.06% APS and 0.12% TEMED Monomer solution not degassed.
Section 8 Product Information and Accessories Mini PROTEAN Tetra Systems Catalog Number Descriptions 165-8000 Mini-PROTEAN Tetra Cell, 10 well, 0.75 mm thickness, complete system includes 5 combs, 5 sets of glass plates, 2 casting stands, casting clamp assembly, sample loading guide, electrode assembly, companion running module, tank, lid with power cables, mini cell buffer dam 165-8001 Mini-PROTEAN Tetra Cell, 10 well, 1.
Catalog Number Descriptions 165-8027 Mini-PROTEAN Tetra Cell and PowerPac HC Power Supply, includes 165-8001 and 164-5052 165-8028 Mini-PROTEAN Tetra Cell and PowerPac HV Power Supply, includes 165-8001 and 164-5056 165-8029 Mini-PROTEAN Tetra Cell and Mini Trans-Blot Module, includes 165-8001 and 170-3935 165-8030 Mini-PROTEAN Tetra Cell (for Ready Gel Precast Gels) and Mini Trans-Blot Module, includes 165-8004 and 170-3935 165-8033 Mini-PROTEAN Tetra Cell, Mini Trans-Blot Module, and PowerPac B
Handcast Gel Accesories and Replacement Parts Catalog Number Descriptions 165-3303 Mini-PROTEAN Casting Stand 165-3304 Mini-PROTEAN Casting Frame 165-3305 Mini-PROTEAN Casting Stand Gaskets, 2 165-3308 Short Plates, 5 165-3310 Spacer Plates with 0.75 mm Internal Spacers, 5 165-3311 Spacer Plates with 1.0 mm Internal Spacers, 5 165-3312 Spacer Plates with 1.
Combs 0.75 mm 1.0 mm 1.
Section 9 Warranty Information The Mini-PROTEAN Tetra cell is warranted for one year against defects in materials and workmanship. If any defects should occur during this warranty period, Bio-Rad Laboratories will replace the defective parts without charge. However, the following defects are specifically excluded. 1. Defects caused by improper operation 2. Repairs or modifications done by anyone other than Bio-Rad Laboratories or their authorized agent 3. Damage caused by accidental misuse 4.
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