3. Isoelectric Focusing (IEF) Protocol

Transcription

1 Proteomics and Mass Spectrometry Facility, BRC, Cornell University Version.0 I. PURPOSE. Isoelectric Focusing (IEF) Protocol This procedure outlines the steps during protein isoelectric focusing that separates proteins according to protein isoelectric points. 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. The IEF procedure should be completed under the temperature controlling. III. PROCESS SUMMARY The first dimension separation procedure involves IPG strip rehydration, sample application, and isoelectric focusing. IV. EQUIPMENT Name Description Special Requirements Eppendorff tube Adjustable Pipettes 0.-0µl, µl Calibrated Pipette tips 00 and 000µl Reswelling Tray Amersham IEF system Multiphor II system Amersham Cooling system MultiTemp III system Amersham Power supply EPS 0 XL Amersham V. MATERIALS LIST Code No Name Supplier Description Milli-Q water In-house Type reagent grade BP69- Urea Fisher T-866 Thio-Urea Sigma ACS Reagent C-946 CHAPS Sigma 6-00 SDS Bio-Rad variety IPG strip Amersham variety IPG Buffer Amersham The ph Compatible with IPG strisp Bromophenol blue (BPB) Bio-Rad 0- Light Mineral Oil (= cover fluid) Fisher scientific NF/FCC; Paraffin oil, light

2 Proteomics and Mass Spectrometry Facility, BRC, Cornell University Version.0 VI. REAGENTS Rehydration solution Final concentration common Urea 7 M Make 7 M Urea/ M Thio-urea/ % CHAPS/BPB stock solution, Thio-urea M aliquot ml and stored at -0 o C CHAPS % for future use. Bromophenol blue Trace DTT 0mM (.08 mg/ml) Add just prior to use IPG buffer % (0µl/ml) Add just prior to use VII. 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.

3 Proteomics and Mass Spectrometry Facility, BRC, Cornell University Version.0. IPG strip rehydration. Prepare the rehydration buffer: Add 0µl of IPG buffer and.8mg DTT into ml of rehydration stock solution and mix very well.. The followed table shows suitable protein amount and rehydration solution volume per IPG strip: Immobiline DryStrip Suitable sample for cup and rehydration loading Silver/Fluorscent Staining Colloidal Coomassie Staining Detection limit: -0ng Coomassie Blue 0 Staining volume /strip Detection limit: Detection limit: 0.-ng 0-0ng µl 7cm ph4-7 L (linear) 4-8 µg 0-0 µg 0-60 µg 7cm ph6- L 8-6 µg -40 µg 0-0 µg 7cm ph-0 L and ph - -4 µg -0 µg 0-60 µg 0 (non-linear) cm ph4-7 L 0-0 µg -40 µg 0-00 µg 00 cm ph6- L 0-40 µg 0-80 µg µg 00 cm ph-0 L 4-8 µg 0-0 µg 0-0 µg 00 cm ph4-7 L -0 µg 0-80 µg 7-40 µg 0 cm ph6- L 0-60 µg 60-0 µg µg 0 cm ph-0 L and µg -40 µg µg 0 8cm ph4-7 L 0-60 µg µg µg 40 8cm ph6-; 6-9 and narrow interval 8cm ph-0 L and -0 4cm ph4-7 L and -7 4cm ph6-9 and narrow interval 4cm ph-0 L and µg µg µg 40-0 µg 0-80 µg 7-40 µg µg µg µg µg µg µg µg µg µg 40. Prepare the reswelling tray: Level the re-swelling tray and slide the protective lid completely off. Ensure that the tray is clean and dry..4 Apply the rehydration (with sample) solution: Mix suitable protein sample with appropriate volume of rehydration and IPG buffer and pipette the mixture to the reswelling slot. Note: expel the solution slowly to the center of the slot, and remove any large bubbles.. Placing the IPG strip: Remove the protective cover from the IPG strip. Position the IPG strip with the gel side down. Gently lift than place the strip down while

4 Proteomics and Mass Spectrometry Facility, BRC, Cornell University Version.0 sliding it back and forth along the surface of the solution. Be careful not to trap bubbles under the IPG strip. Note: Dry IPG strip is easily moistened. Take dry IPG strip from freezer just prior use. Don t allow IPG strip set at room temperature more than 0 min..6 Overlay each IPG strip with. to ml of IPG Cover Fluid to prevent evaporation and urea crystallization..7 Slide the lid onto the reswelling tray and allow the IPG strips to rehydrate at room temperature overnight. (A minimum of 0 hours is required for rehydration). Prepare the immobiline DryStrip Kit. Clean all components of the Immobiline DryStrip Kit. Usually clean the Kit is cleaned after use but it is necessary to check it before using it.. Confirm electrical connections on Multiphor II. Position the cooling plate on the Multiphor II unit and ensure that the surface is level. Set the temperature on MultiTemp III Thermostatic Circulator to 0 o C. This step is only for first time using Multiphor II..4 Pipette about 0 ml of IPG Cover Fluid onto the cooling plate and position the Immobiline DryStrip tray on the cooling plate so the red electrode connection of the tray is positioned at the top of the plate near the cooling tubes. Remove any large bubbles between the tray and the cooling plate to increase the contact cooling surfer. If using IEF system very often, the DryStrip tray can be set on the cooling plate all the time.. Pour about -0 ml of IPG Cover Fluid into the immobiline DryStrip tray. Place the DryStrip aligner into the tray on top of the IPG Cover Fluid slowly and remove the large bubbles between the two surfaces. Avoid getting IPG Cover Fluid on top of the aligner at this point..6 Prepare electrode strips: Cut two IEF electrode strips to a length of 0mm. Moisten each strip with 0. ml Milli-Q water and dump the electrode strips on filter paper. The strips should be damp too much moisture within the strips will cause uneven focusing of the sample. Set aside. Prepare for electrophoresis. Remove the IPG strip from its slot in the Re-swelling Tray carefully. Hold one end of IPG strip with forceps and rinse the IPG strip with Milli-Q water completely to remove excess rehydration solution and thus prevent formation of urea crystals on the gel surface during IEF. Place the IPG strip on its edge on a

5 Proteomics and Mass Spectrometry Facility, BRC, Cornell University Version.0 damp filter paper for several seconds to drain excess moisture. Note: Avoid contact between the gel surface and the filter paper.. Immediately transfer the rehydrated IPG strip to adjacent grooves of the aligner in the immobiline DryStrip tray. Place the strips with the point (acidic) end at the top of the tray near the red electrode (anode) and gel side face up. The blunt end should be at the bottom of the tray near the black electrode (cathode). All strips must have the same length.. Place electrode strips across the cathode and anodic ends of the aligned IPG strips. The electrode strip must at least partially contact the gel surface of each IPG strip..4 Align each electrode over an electrode strip, ensuring that the marked side corresponds to the side of the tray giving electrical contact. Press electrode down to contact the electrode strips.. Cover the IPG strips with 80 ml of DryStrip Fluid to prevent evaporation and urea crystallization.. Isoelectric focusing This method of focusing is conducted at hight voltages and low currents due to low ionic strength within the strips. During IEF, the current decreases while the voltage increases. A low initial voltage minimizes sample aggregation and allows the parallel separation of samples with differing salt concentrations. The program used is dependent on the size of strip being focused and the quantity of protein on the strip. 4. Before starting the running program, the Multiphor II cover has to be seated completely. 4. The protocol given in below table is suitable for first-dimension isoelectric focusing of protein samples in Multiphor II. (Check parameter always with vendor s instruction and add always h to the last step of recommended time.) IPG strip Phase Length ph range 7cm ph4-7 L 7cm ph6-l;-0l and -0 cm ph4-7 L Voltage (V) Current (ma) Power (W) Duration (h:min) 0:0 :0 0:-:0 :-:00 0:0 :0 0:-:0 :0-: 0:0 :0 :0-:0 Vh (recommended)

6 Proteomics and Mass Spectrometry Facility, BRC, Cornell University Version.0 cm ph6-l and -0L cm ph4-7 L cm ph6-l;-0l and -0 :0-: :0 00 : :4-: :0-4: cm ph4-7 L 8cm ph6-l;-0l and -0 4cm -0 L 4cm ph4-7l;-7 and -0 4cm ph6-9l 4cm ph.-4.; ; and.-6.7l :0 :0 :4-4:0 :-:0 0:0 :0 :0-4:0 4:40-6:00 0:0 :0 :40-7:40 7:0-9:0 0:0 :0 4:0-6:0 6:0-7:0 0:0 :0 7:40-0:40 9:0-:0 0:0 :0 :00-6:0 :0-7:4 0:0 :0 6:0-4:0 7:0-6;0 0:0 :0 :00-7:40 :0-9; Connect the leads on the lid to the power supply. Ensure that the current check on the EPS 0 XL power supply is switched off (in the set up menu). Begin IEF. 4.4 As isoelectric focusing proceeds, the dye migrates toward the anode. Note: The dye front leaves the IPG strip well before focusing is complete, so clearing of the dye is no indication that the sample is focused. 4. After IEF proceed to the second-dimension separation immediately or store the IPG strips at -40 to -80 o C in equilibration tube individually.