ReadyPrep™ 2-D Starter Kit Instruction Manual Catalog Number 163-2105 For technical service call your local Bio-Rad office or in the U.S. call 1-800-4BIORAD (1-800-424-6723) On the Web at http://www.discover.bio-rad.
Table of Contents Section Section Section Section Section Section 1 2 3 4 5 6 Introduction ......................................................................1 Kit Components............................................................1-2 Storage ............................................................................2 Instructions for Use ..........................................................3 Appendix........................................................................
Section 1 Introduction The ReadyPrep™ 2-D starter kit was designed as a single-use kit to familiarize first-time users with the utilization of the Bio-Rad PROTEAN® IEF cell and ReadyStrip™ IPG strips. The kit provides all the reagents necessary, including a protein control, to successfully perform two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The kit contains sufficient reagents to perform 2-D PAGE of at least 6 samples with any of the three SDS-PAGE formats Bio-Rad offers.
Overlay Agarose. One bottle containing 50 ml of 0.5% low melting point agarose in 25 mM Tris, 192 mM glycine, 0.1% SDS, and a trace of Bromophenol Blue. Instruction Manual. One. Note: CHAPS is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent. Bio-Lyte® 3/10 ampholytes is a mixture of carrier ampholytes, pH 3–pH 10.
Section 4 Instructions for Use Introduction This 2-D starter kit allows you to successfully separate a complex protein extract by 2-D PAGE using IPG strips and the Bio-Rad PROTEAN IEF cell. The buffer formulations as well as the following protocol have been optimized for the E. coli protein sample included in the kit. Before beginning, decide which length IPG strips you wish to use and how many you wish to focus. The kit contains sufficient reagents for the separation of the E.
7 cm Day 1 11 cm 17 cm Rehydration Setup 30 min Rehydration Setup 30 min Rehydration Setup 30 min IPG Rehydration 12 hours IPG Rehydration 12 hours IPG Rehydration 12 hours Isoelectric Focusing 5 hours Isoelectric Focusing 5.
4.1 4.2 Sample Preparation 1 Remove the bottle of rehydration/sample buffer, the bottle of nanopure water and the vial of E. coli protein sample from the kit. All 3 bottles have a GREEN cap. 2 Remove the desired number of pH 4-7 ReadyStrip IPG strips from the -20ºC freezer and set aside. (Six strips are recommended: 2 to stain after IEF, 4 to be used for seconddimension SDS-PAGE).
Fig. 2. Sample loading of rehydration/equilibration trays. Pipet the sample along the back edge of the tray channel except for about 1 cm at each end. Note the even distribution of the sample along the edge of the channel. The figure shows the last of six samples being pipeted in the tray. 2. Repeat this process for the remaining samples by pipetting the indicated volume of sample into adjacent channels. It is best to place samples on both sides of the tray as in Figure 2.
Gently place the strip gel side down onto the sample as illustrated in Figure 4. The “+” and “pH 4-7” should be legible and positioned at the left side of the tray. Take care not to get the sample onto the plastic backing of the strips as this portion of the sample will not be absorbed by the gel material. Also take care not to trap air bubbles beneath the strip.
4 5 4.3 Overlay each of the strips with 2 to 3 ml of mineral oil to prevent evaporation during the rehydration process. Add the mineral oil slowly, by carefully dripping the oil onto the plastic backing of the strips while moving the pipet along the length of the strip. Mineral oil prevents evaporation of the sample during rehydration, thus preventing precipitation of the urea. IPG strips can be left to rehydrate for up to 1 hr before adding the oil.
Fig. 6 Wetting the electrode wicks with water. Fig. 7 Draining the oil. Fig. 8 Placing the ReadyStrip gel side down in the focusing tray.
Draining the oil essentially washes the outside surface of the gel, removing unabsorbed protein and resulting in reduced horizontal streaking. If desired, the disposable rehydration/equilibration tray can be cleaned, dried, and used to store strips after the completion of the first-dimension run. 5 Remember to observe the correct polarity during the transfer of strips into the focusing tray.
4.4 Completion of IEF. 1. When the electrophoresis run has been completed, remove the IPG strips from the focusing tray and transfer them gel side up into a new or clean, dry disposable rehydration/equilibration tray which matches the length of the IPG. Hold the strips vertically with the forceps and let the mineral oil drain from the strip for ~5 seconds before transfer. Maintain the IPG strips in the same order as in the focusing tray.
Fig. 9. Isoelectric focusing of E. coli protein sample in 7 cm, 11 cm, and 17 cm IPG strips followed by staining with IEF gel staining solution. 6 4.6 Figure 9 shows the expected pattern when IPG strips of 17 cm, 11 cm, and 7 cm are stained with IEF gel staining solution (catalog # 161-0434) and destained with Coomassie Brilliant Blue R-250 destaining solution (catalog # 161-0438). Preparations Needed Before Beginning IPG Equilibration & SDS-PAGE.
a Remove the kit from the refrigerator and unpack the 2 bottles of equilibration buffer I (WHITE or SILVER caps), the 2 bottles of equilibration buffer II (RED caps), the bottle of 30% glycerol (CLEAR cap), the 2 bottles of iodoacetamide (RED caps), and the bottle of overlay agarose (CLEAR cap). b Remove the crimp and open the two bottles of equilibration buffer I and equilibration buffer II.
6. Using Table 3, add the indicated volume of complete equilibration buffer II (containing iodoacetamide) to each strip. 7. Return the tray to the orbital shaker for 10 minutes. 8. During the incubation, melt the overlay agarose solution in a microwave oven using the following method. a Loosen the cap of the bottle of overlay agarose and place the bottle in the center of a microwave oven. b Microwave on high 45 – 60 seconds until the agarose liquifies.
3. Remove an IPG strip from the disposable rehydration/equilibration tray and dip briefly into the graduated cylinder containing the 1X Tris/glycine/SDS running buffer, as shown in Figure 10. Lay the strip gel side up and onto the back plate of the SDS-PAGE gel above the IPG well (Figure 11). Repeat this process for any remaining IPG strips. Fig. 11. Placing the ReadyStrip gel side up on the back plate of the SDS-PAGE gel. 4.
5 Using the forceps, carefully push the strip into the well as shown in Figure 13, taking care not to trap any air bubbles beneath the strip. When pushing the IPG strips with the forceps be certain the forceps are pushing on the plastic backing to the strip and not the gel matrix. Fig. 13. Sliding ReadyStrip into IPG well filled with melted overlay agarose. 6. Stand the gel(s) vertically by placing them in the gel box (Criterion system) or in a test tube rack.
4.9 STAINING. The 2-D starter kit was designed so that sufficient protein sample loaded in the IEF dimension readily stain second-dimension SDS-PAGE gel with widely available Coomassie Blue stains such as Bio-Rad’s Bio-Safe Coomassie stain and Coomassie Brilliant Blue R250 stain. These staining protocols are rapid and easy to perform so that the 2-D electrophoresis results can be quickly assessed. A description of each staining protocol is provided below. 1. BioSafe Coomassie Stain.
4.10 1 Imaging. To preserve the gel images, the destained gels can be imaged on a densitometer such as Bio-Rad’s GS-800 calibrated imaging densitometer (catalog # 170-7980). Alternatively, gels can be equilibrated for at least 30 minutes in gel drying solution (catalog # 161-0752) and dried. Fig. 14 A. Ready Gel stained with Bio-Safe stain. Fig. 14 B. Criterion stained with Bio-Safe stain.
Fig. 14 C. PROTEAN Ready Gel stained with Bio-Safe stain.
Figure 15 A. Ready Gel stained with Coomassie Blue R-250 stain. Fig. 15 B. Criterion stained with Coomassie Blue R-250 stain.
Figure 15 C. PROTEAN Ready Gel stained with Coomassie Blue R-250 stain.
Section 5 Appendix A Buffer and Reagent Preparation To avoid introducing experimental error when using this kit, we suggest using premixed buffers and stains. These can be found in section 6, Product Information. 1X Tris/glycine/SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) Tris base 3.03 gm Glycine 14.4 gm SDS 1.0 gm Water to 1 L, do not adjust pH Coomassie Brilliant Blue R-250 stain solution (0.1% Coomassie Blue R-250 in 40% MeOH, 10% HOAc) Coomassie Blue R-250 1.
4. - The first line displays the step number, slope and voltage Select HRS:MIN (default) Enter time of 20 min. Press NEXT 5. - Select ADD (default) - Enter Step 2 - Press NEXT S 01 HRS:MIN ENTER TIME: 250 V vhours 00:20 NEXT ADD inset delete > ENTER STEP # > > > 2 > NEXT METHOD COMPLETE > > 6. - Enter step 2 final voltage 7 cm: 4,000 V; 11 cm: 8,000 V; 17 cm: 10,000 V) - Select LINEAR ramp.
11. - Select METHOD COMPLETE ADD inset delete ENTER STEP # NEXT METHOD COMPLETE > > > > 12. - The save method screen is displayed when the name entered “Ecoli” > is displayed SAVE METHOD? YES no > - Select YES NEXT > - Press NEXT 13.
Section 6 Product Information Catalog Number Product Description 2-D Starter Kit accessories 163-2105 163-2106 163-2107 163-2108 163-2109 163-2110 163-2111 ReadyPrep 2-D Starter Kit ReadyPrep Rehydration/Sample Buffer, 1 bottle, 10 ml ReadyPrep Equilibration Buffer I, 1 bottle, 20 ml ReadyPrep Equilibration Buffer II, 1 bottle, 20 ml Iodoacetamide, 30 gm ReadyPrep E. coli Protein Sample, 1 vial, 2.
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