Protocol for 2D-E. Protein Extraction

Transcription

1 Protocol for 2D-E Protein Extraction Reagent 1 inside the ReadyPrep TM Sequential Extraction kit (in powder form) 50ml of deionized water is used to dissolve all the Reagent 1. The solution is known as R1 and is stored under - 20 c Reagent 2 inside the ReadyPrep TM Sequential Extraction kit (in powder form) 6ml of deionized water is used to dissolve all the Reagent 2. The solution is known as R2 and is stored under - 20 c 1. Prepare around 20mg of tissue in eppendorf 2. For each sample, add the following materials: 0.1ml R1 0.4µl of (RNase A + DNase I) 10ul 10X complete proteose buffer 10ul 10X DNase I buffer 3. Add 0.1ml of R1 and grind for 1.5 minutes 4. Centrifuge at maximum speed for 10 minutes at 4 c 5. Store the Supernatant in a new tube S1 6. For each ml of R2 (normally require 50-70µl R2), add 10µl reducing agent TBP together in the fumehood and mix it well 7. Solubilize the pellet in R2 by grinding it for 1.5 minutes 8. Centrifuge at maximum speed for 10 minutes at 4 c 9. Store the supernatant in a new tube S2 10. Centrifuge S1 and S2 at maximum speed for 60 minutes at 4 c 11. Use supernatant of S2 to run gel Protein Quantification 1. Supernatants of S1 and S2 (from protein extraction 2. 6 standard tubes 3. 2 sample tubes for each sample 4. 5X Protein Assay Dye Reagent Concentrate (450ml) 5. Protein assay Standard II: Lyophilized Bovine Serum Albumin (BSA) (20ml) Preparation 1. Rehydrate BSA with 20ml of nanopure water to obtain a solution of 1.37mg/ml

2 2. Dilute 3ml of 5X Protein Assay Dye Reagent Concentrate solution to a total volume of 15ml (1X) (1ml per tube) Preparation of Standard tubes 1. Six standard tubes are to be at the start: Tube 0: 25ml BSA (1.37 mg/ml) Tube 1: 25ml nanopure water + 25ml BSA (mix well) Tube 2: 25ml nanopure water Tube 3: 25ml nanopure water Tube 4: 25ml nanopure water Tube 5: 25ml nanopure water 2. Mix well the contents of tube 1. Pipette 25ml of the solution in tube 1 and put it into tube Mix well the contents of tube 2. Pipette 25ml of the solution in tube 2 and put it into tube Mix well the contents of tube 3. Pipette 25ml of the solution in tube 3 and put it into tube Mix 10µl of each of the solution inside Tubes 1 to 6 with 1ml of 1X Assay solution into 6 new tubes Preparation of Sample tubes 1. Label 2 tubes (S1 and S2) for each sample 2. Dilute samples by 20X, i.e. for each of the S1 and S2, 19µl of Nanopure water and 1µl of sample lysate, mix thoroughly 3. Mix 10µl of each of the solution inside all S1 and S2 with 1ml of 1X Assay solution into new tubes 4. Stand for 5 minutes in room temperature. 5. Measure OD595

3 Isoelectric Focusing IPG Buffer (3-10, 4-7, 6-11) 1% Bromophenol Blue 40mM DTT (0.006g/ml) 8M Urea /w 2% CHAPS / 7M Urea + 2M thiourea /w 4% CHAPS Sample loading 1. Prepare IEF Buffer with following constituent (per 1ml): a g DTT b. 5ul IPG buffer c. 2ul bromophenol blue d. 8M Urea /w 2% CHAPS or 7M Urea /w 2M thiourea and 4% CHAPS (Make up to 1ml) 2. Add 25-50ug protein to 200ul (11cm) or 450ul IEF buffer (24cm) 3. Add the sample mixture to strip holder 4. Place IPGphor dry strip on top of sample mixture, prevent bubbles 5. Overlay with equal amount of IPG cover oil, prevent bubbles and re-assure coverage. 6. Cover the strip holder IEF 7. Place the strip holder onto IEF machine, align with corresponding sign 8. Set IEF as following a. 50uA per strip b hours 30V c. 1 hr 100V d. 1 hr 500V e. 1 hr 1000V f. 2 hrs (5hrs) 8000V/11cm (24cm)* 9. After completion, strip can be stored in -80C *Time varies and target is 20000VHrs for 11cm and VHrs for 24cm

4 Second dimension Equilibration (EQ) Buffer (50mM tris ph8.8 /w 6M urea, 30% glycerol, 0.002% bromophenol blue) DTT (0.1g/10ml EQ buffer) Iodoacetamide (IAA, 250mg / 10ml EQ buffer) Tris/Glycine (Cathode buffer without SDS) 100X Anode buffer (262.8ml diethanolamine, 155g acetic acid per 500mL) 10X Cathode buffer (144g tris g glycine + 50ml 20% SDS per 1L) Equilibration of strip 1. Incubate strip with 10ml of EQ buffer + DTT for 15 min, r.t. 2. Equilibrate strip with 10ml of EQ buffer + IAA for 15 min, r.t. 3. Rinse strip with Tris/Glycine Strip loading 1. Place strip on top of the SDS PAGE gel, add a little Tris/glycine to fill gaps and remove excessive bubbles. 2. Remove excessive tris/glycine (form bubbles during run) by squeezing the strip gently. 3. Overlay with 0.5% agarose in cathode buffer with 0.002% bromophenol blue 2D PAGE 1. Fill tank with 5L 1X anode buffer 2. Place gels into tank 3. Add 1L 1X cathode buffer on top 4. Start run (24cm gel 2.5W/gel 30min and 10mA O/N; 11cm gel 25mA 5 hrs)

5 Silver Staining (Refer to kit Amersham ) Fixing solution, 500ml per gel (200ml EtOH + 50 ml acetic acid per 500ml) Sensitizing solution, 250ml per gel (75ml EtOH, 10ml 5% (w/v) sodium thiosulfate and 17g sodium acetate) Silver staining solution, 250ml per gel (25ml 2.5% AgNO 3 per 250ml) Developing solution, 250ml per gel (6.25g NaCO 3, 100ul 37% (v/v) formaldehyde per 250ml) Stopping solution, 250ml per gel (3.65g Na 2 EDTA per 250ml) 1. Fix gel with fixing solution (200ml EtOH, 50ml acetic acid per 500ml) for 1hour x 2 times 2. Sensitize gel with 250ml sensitizing solution (75ml EtOH, 10ml 5% sodium thiosulfate and 17g sodium acetate per 250ml) for 1 hour 3. Wash gels with NANOPURE-water for 10 min x 4 times 4. Incubate gels in 250ml silver nitrate (25ml 2.5% w/v silver nitrate per 250ml) for 1 hr 5. Wash gels with NANOPURE-water for 1 min x 4 times 6. Develop color with 250ml developing solution containing 6.25g sodium carbonate, 100ul 37% formaldehyde 7. Stop developing by adding 250ml of Na2EDTA (3.65g/250ml) 8. Wash gel with water for 10min 9. Scan gel image with densitometer (PDQuest) at 400dpi

6 Spot picking before performing Mass Spectrometry 15mM of K 3 Fe(CN) 6 of MW (i.e g in 1 ml) 50mM of Na 2 S 2 O 3 of MW (i.e g in 1 ml) 50mM of NH 4 HCO 3 of MW (i.e g in 1 ml) 20mM of NH 4 HCO 3 of MW (i.e g in 1 ml) Destain 1. Prepare 1ml of solution 1 (containing 15mM of K 3 Fe(CN) 6 and 50mM of Na 2 S 2 O 3 ) 2. Add 100ul to each gel spot 3. Vortex and spin down for 3 seconds (or centrifuge down the gel) 4. Stand in room temperature in the dark for 20 minutes Wash 1. Remove the destain solution carefully 2. Add 100ul H 2 O, stand in room temperature in the dark for 20 minutes. Repeat this step for 3 to 4 times. Equilibrium 1. Prepare 50mM of NH 4 HCO 3 2. Add 100ul of the above into each tube, stand in room temperature in the dark for 10 minutes 3. Remove the solution 4. Add 100ul ACN, stand in room temperature in the dark for 5 minutes. Repeat this step for 2 times. 5. Speed-vacuum for 5 minutes Trypsin in-gel digestion 1. Trypsin working concentration: 1µg/µl 2. Add 10-20ul of trypsin to each tube 3. Incubate at 37ºC for overnight Elute 1. Prepare TFA + ACN containing 0.1% TFA, 0.5ml ACN and 0.5ml H 2 O 2. Add 35ul each 3. Vortex at maximum speed for one minute, spin down in ultrasonic shaker to increase protein yield, stand at room temperature for 20 minutes 4. Transfer all solution to a new tube (~50 60ul for the first time) 5. Repeat steps 2 to 4 again 6. Speed-vac for hour, store dried protein at -80ºC.

7 Preparation process before performing Mass Spectrometry 0.1% Trifluoroacetic acid (TFA) 0.1% TFA + 50% ACN (0.5ml CAN ml H 2 O + 1ul TFA) 50% ACN (in H 2 O) Zip Tip C 18 Pipette, tips, 2ul & 10ul 1.5ml tube (store waste) PCR tubes (aliquot TFA, TFA+ACN, ACN) CHCA matrix Sample loading 1. Add 10ul 0.1% TFA to powder, vortex and spin down 2. Ultrasonic in water bath for 1 min 3. Wet Zip Tip by 10 ul 50% ACN until use for around 15 minutes Equilibrium 1. Wash the tubes with 10ul of 0.1% TFA 3 times Binding sample 1. Re-suspend the sample with Zip Tip for 10 times 2. Transfer 10ul of sample to the binding tube (Optional) Washing 1. Wash the sample with 10ul TFA for 3 times Elute 1. Elute peptide with 2ul of 0.1% TFA /w 50% ACN 2. Pipette up and down for 10 times 3. Transfer the final product to the MALDI- plate 4. Transfer 1ul (TFA + ACN) to chips using new pipette tip 5. Add 1ul CHCA above the sample spot 6. Mark the log sheet 7. Air dry