4. The Second-dimensional SDS-PAGE (vertical) Protocol

Transcription

1 4. The Second-dimensional SDS-PAGE (vertical) Protocol I. PURPOSE This procedure outlines the steps that must be carried out in the seconddimension SDS-PAGE using vertical system. II. ENVIRONMENT All work must be done in a clean area. Special care should be taken to prevent contamination of the samples with keratin; i.e., wear gloves and Lab coat at all times when handling tubes, reagents and equipment that will come in contact with samples. This procedure should be completed under the temperature control which will improve gel-to-gel reproducibility. III. PROCESS SUMMARY The protocol includes preparation of the SDS-acrylamide gel, equilibration of the IPG strip(s) in equilibrium buffer (for reduction and alkylation), manipulation of the equilibrated IPG strip on SDS-gel, and running electrophoresis. IV. EQUIPMENT Name Description Special Requirements Adjustable Pipettes µl, µl Calibrated Pipette tips 200 and 1000µl Hoefer minive Gel size: 6x8 cm Amersham Hoefer SE 600 Gel size: 18x16 cm Amersham Ettan DALTtwelve System Gel size: 21.5x25 cm Amersham Ettan DALTsix System Gel size: 20.5x25.5 cm Amersham MultiTemp III system Cooling system (Hoefer SE 600) Amersham EPS 601 power supply Power supply (Hoefer SE 600) Amersham Lab glass ware For preparing solution Equilibration tube Amersham

2 V MATERIALS LIST Code No Name Supplier Milli-Q water In-house VW SDS (MW: g/mol) General Storage Bromophenol blue (BPB) Bio-Rad Low molecular weight std Amersham Agarose Amersham BP152-5 (5kg) Tris-Base (MW: g/mol) Fisher scientific BP318-5 (5kg) Glycine (MW: g/mol) Fisher scientific EC-890 ProtoGel National Diagnostics Acrylamide N,N - Acros organics Methylenebisacrylamide (37.5:1; 40% mix solution in water) Ammonium persulphate Amersham TEMED Bio-Rad PlusOne Dithiothreitol (DTT) Amersham Iodoacetamide 98% (IAM) Acros organics V. REAGENTS 1.5M Tris-Cl ph8.8 Tris (MW ) 182g Adjust to ph 8.8 and store at 4ºC 6 M HCI to ph 8.8 About 50ml Deionized water To 1000ml SDS equilibration buffer Final concentration 1.5M Tris-Cl ph mm Urea 6 M Make stock solution and aliquot 20 ml and Glycerol 30% stored at -20 o C. SDS 2% Bromophenol blue trace DTT 100 mg per 10 ml Add just prior to use Iodoacetamide 98% (IAM) 250 mg per 10 ml Add just prior to use Electrophoresis running buffer (10x) Tris-Cl 250 mm Stock this 10x electrophoresis running buffer Glycine 1.92 M at room temperature. Dilute it 10 times before using. SDS 1% 10% Ammonium persulfate solution (APS) Ammonium Persulfate 100mg Prepare fresh Deionized water 1ml 1% Agarose sealing solution Agarose 1g Heat the solution until the agarose is

3 Electrophoresis buffer 100ml completely dissolved Displacing solution (for DALT only) 1.5M Tris Cl ph8.8 25ml Should be made fresh; stored solution may Glycerol 50ml support microbial growth. Bromophenolblue 2mg Deionized water 25ml SDS 10% Overlay solution ( water saturated butanol) n or t-butanol 50 ml Shake and use top phase to overlay the Deionized water 5 ml monomer solutions VI. PROCEDURE NOTE: All equipment and materials that come into contact with gel samples must be clean and free of keratin contamination. Minimize handling and manipulations of samples. 1. Preparing SDS Gel: 1.1 Select the gel percentage: Single percentage offer better resolution for a particular MW window. A commonly used second-dimension gel for 2-D electrophoresis is a homogeneous gel containing 12.5% total acrylamide. The appropriate percentage gel is selected according to the range of separation desires. The table recommended acrylamide concentrations for protein separation: % Acrylamide in resolving gel Separation size range (MWx10-3 ) Single percentage: 5% % % % % Gradient: 5-15% % % Select the gel thickness: Either 1.0 mm or 1.5 mm

4 1.2 Casting homogeneous : SE 615 multiple caster can cast up 10. DALTtwelve caster can cast up 14. Be sure, that gel casters, spacers, separator sheets and cassettes or glass plates are perfectly clean and dry. Place the cassettes alternating with separator sheets into the caster. Block the spare space with blank cassettes when less than 12 (DALTtwelve caster) or less than 10 (SE 615 caster) should be prepared. Level the gel caster horizontally. 1.3 Preparing monomer solutions and casting homogeneous : 10 % 12.5% 15% Acrylamide stock 40%ig 25 ml 31 ml 37.5 ml 1.5M Tris-Cl ph ml 25 ml 25 ml 10% SDS 1 ml 1 ml 1 ml Water ml 36 ml TEMED 33µl 33µl 33µl 10% Ammonium persulfate 500µl 500µl 500µl Total volume 100 ml 100 ml 100 ml caster Hoefer minive Hoefer SE 600 Hoefer SE 615 Daltsix Dalttwelve Number of Acrylamide solution volume 850 ml 100 ml For 1.5 mm thick 60 ml per gel for 1.5mm thick 450 ml 500 ml for 1mm thick TEMED is added together with the stock solutions, it cannot catalyses the polymerization without ammonium persulfate. The ammonium persulfate should not be added before everything is in place, because there are only about 10 minutes left to pour the gel until it starts to gel. Pour the gel solution into the funnel using a 50 ml pipette, avoiding air bubbles. Stop pouring when the level has reached 1 cm below the upper edges of the casting cassettes.

5 Immediately pipette 1 ml overlay solution to each cassette. Let the gel polymerize for one hour (It is important to use the same volume for each cassette.). Inspect each gel cassette for eventual air bubbles. Gels with air bubbles should not be used. Wrapp the gel cassettes into wet paper towels and put it in a plastic bag. Store the usable in a cold room or refrigerator. 2. IPG strip equilibration 2.1 Take the equilibration buffer out of the freezer and prepare 1% agarose-solution. Heat the agarose solution on a heating stirrer until agarose gel is completely melted. The equilibration procedure also serves as reduction and alkylation for IPG strips. The table recommends suitable equilibration volume: IPG strip 13 or under 13cm 18cm 24 cm Equilibration buffer* 10ml 15ml 20ml * 40 ml aliquots are frozen and have to be split, add DTT and IDA always freshly Reduction Add DTT-containing equilibration buffer (100mg DTT/10ml buffer) to each IPG strip individual equilibration tube, place it on a rocker and equilibrate 15 min before pouring the buffer out Alkylation Add Iodoacetamide-containing equilibration buffer (250mg IAM/10ml buffer) to each IPG strip individual equilibration tube, place it on a rocker and equilibrate 15 min before pouring the buffer out. 3. Applying the IPG strip 3.1 Rinse the IPG strip with running buffer and place the strip with acidic end (+) to the left and plastic backing against one of the glass plates. With a thin plastic ruler, gently push the IPG strip down so that the entire lower edge of the IPG strip is in contact with the top surface of the slab gel. Ensure that no air bubbles are trapped between the IPG strip and the slab gel surfaces or between the gel backing and the glass plate. Note: If using 24 cm strip it has to be cut on the (-) end after placing against the glass plate and before pushing down. 3.2 Optional: If you want to apply a molecular weight marker proteins place sample application piece next to the acidic end.

6 3.3 Seal the IPG strip with 1% agarose gel. Allow the agarose to cool down until the tube can be held by fingers (60ºC) and then slowly pipette the amount required to seal the IPG strip in place. Pipetting slowly avoids introducing bubbles. 3.4 Load molecular weight marker proteins (Blue range + 10µl water) after inserting the precast gel cassettes into Hoefer 600 electrophoresis system and carefully pulling out the sample application piece. 4 SDS electrophoresis 4.1 Prepare electrophoresis running buffer and turn the cooling system on. The recommended temperature for SE 600 is 15ºC, for DALTsix is 5ºC and for DALTtwelve depends on running conditions. Electrophoresis buffer for Ettan DALTsix Gel thickness Anodic buffer (lower chamber) Volume [l] 1 mm 1xSDS electrophoresis buffer Cathodic buffer (upper chamber) 4.3 2xSDS electrophoresis buffer Volume [l] Preparing the electrophoresis unit DALTsix: Follow the instructions in Manual p12 DALTtwelve: Insert the precast gel cassettes. When the electrophoresis buffer has reached the desired temperature, insert loaded gel cassettes, push blank cassette inserts into any unoccupied slots. When 12 slots are occupied, the buffer level should be slightly below the level of the gaskets (0.5 cm). Note: Cassette insertion is aided by spraying the gel cassettes with a mist of SDS electrophoresis buffer prior to insertion.

7 4.3 Electrophoresis conditions: Electrophoresis system step Current (ma/gel) Power(w/gel) Duration(h:min) Hoefer minive 1.5 mm-thick : : mm-thick : :30 Hoefer SE mm-thick : : mm-thick : :00 DALTsix 1.0 mm-thick Phase (step) Temperature Power Duration (h:min) 1 5ºC (Yong) or 25 C 30W for 6 5 W per gel 30 min 600 V; 400 ma W for 6 7h 800 V; 800 ma (17) max 100 W per gel DALTtwelve 1.0 mm-thick Phase Temperature Power Duration (h:min) 1 28ºC 5 W per gel min W Max Approximately 5 h for ºC 5 W per gel min W max Approximately 6 h for ºC 5 W per gel min W max Approximately 8 h for ºC 5 W per gel min 2 80 W max Approximately h for ºC 1-2 W per gel min 2 Overnight The Table lists the recommended conditions for the Hoefer minive; SE600 and Ettan DALTsix and Ettan DALTtwelve systems.

8 Electrophoresis is performed at constant current (or constant power for DALTtwelve) in two steps. During the initial migration and stacking period, the current is approximately half of the value required for the subsequent separation. Do not cool SDS below 10ºC. For the smaller system (such as mini-gel), cooling is optional. The temperature control improves gel-to-gel reproducibility, especially if the ambient temperature of the laboratory fluctuates significantly. 4.4 Stop electrophoresis when the dye front line is approximately 1mm from the bottom of the gel. 4.4 After electrophoresis, remove from their gel cassettes in preparation for fixing and staining and cut a small piece of gel from the upper left corner (acidic side) off.