2D gel Protocol

1. Lysis and Protein Extraction from cells

Prepare cell lysates with Trizol extraction by following Kathleen Lyonss protocol: AfCS Procedure Protocol PP00000155, Version 1, 05/12/03 (Ref.1). This preparation method provides total cellular protein samples that are free of contaminating nucleic acids and free of protease activity. The protein extracts are used for analysis of proteins by two-dimensional gel electrophoresis.

2. Determining Protein Concentration of cell lysates

Perform a Bradford protein assay (Bio-Rad Protein Assay Instructions come with the Protein Assay Kit I, Catalogue No. 500-0001 (Ref.2)) to determine protein concentration.

3. First dimension Separation: Isoelectric Focusing (IEF)

This protocol uses a 13 cm strip as an example This protocol is based on that found in the handbook prepared by Amersham Biosciences: 2-D Electrophoresis using immobilized pH gradients Principles and Methods 80-6429-60 (Ref.3) 1) Decide what size of IPG strip you will use for your experiment and how much of your sample to use. The following table provides a range for Sample loading onto a IPG strip. Table 1: Suitable sample loads for silver and Coomassie staining Immobiline Dry Strip
7cm

pH
4-7 6-11 3-10, 3-10 NL 4-7 6-11 3-10 L 4-7 6-11 3-10, 3-10 NL 4-7 6-11, 6-9, narrow interval 3-10, 3-10 NL 4-7, 3-7 6-9, narrow interval 3-10, 3-10 NL

Suitable Sample Load (ug of protein) Silver stain Coomassie stain

4-8 8-16 2-6 10-20 20-40 4-8 15-30 30-60 8-15 30-60 60-120 15-30 45-90 80-170 20-40 20-120 40-240 10-60 50-300 100-600 20-120 75-450 150-900 40-240 150-900 300-1500 75-450 200-1300 400-2000 100-600

11cm

13cm

18cm

24cm

2) Prepare the strip holder(s) corresponding to the IPG strip length chosen for the experiment. Handle the ceramic strip holder with care, as they are fragile.

3) Wash strip holder: Rinse each strip holder with ddH2O followed by adding a few drops of Ettan IPGphor Strip Holder Cleaning Solution. Use a toothbrush and vigorous agitation to clean the strip holder. Rinse well with ddH2O. Thoroughly dry the strip holders with lint-free tissue prior to use. 4) Prepare fresh rehydration solution For example: thaw 945uL rehydration stock (stock in the 80C freezer), add 50uL of 1M DTT and 5uL of IPG buffer solution mix well. 5) Prepare the sample and rehydration solution mixture: add appropriate volume of rehydration solution into the sample to make up the total volume indicated in the following table Table 2. Sample and rehydration solution mixture volume per IPG Strip IPG Strip length (cm) 7 11 13 18 24 Total volume per strip (uL) 125 200 250 340 450

For example: a sample is determined to have 5.7 ug/uL total protein. Only 150ug of this protein sample will be run on a 13cm pH 4-7 strip. Therefore, 26.3uL of this sample plus 223.6 uL of rehydration solution will be mixed up to make 250uL of total volume. This sample will be used for SYPRO ruby staining. 6) Distribute the rehydration-sample solution mixture evenly in the strip holders. Be sure to record the boat number with the sample name. 7) Remove the protective cover from the IPG strip starting at the positive (acidic, pointed or arrow) end. Position the IPG strip in the reswelling holder with the gel side down and the positive end of the strip against the sloped end of the slot. 8) Overlay each IPG strip with DryStrip Cover Fluid to minimize evaporation and urea crystallization. This amount is variable (eg. 300-400uL for 7cm and 700800uL for 13cm strips). 9) Run your IPG strip on the Ettan IPGphor II. Align each strip with the strip length markers present on the IPGphors. Put IPG strip positive on IPGphor positive side. Place the internal cover over the ceramic holders and close the lid. Turn on the machine. Use the up and down cursors to select which protocol you need (check the following table for protocols). Use the up and down cursors to select how many strips you will be running (up to 6?). Table 3. Guidelines for Ettan IPGphor with rehydration loading/IEF for Immobiline Dry Strips. Voltage step and hold mode, 50uA/IPG strip, 0.5% IPG buffer, 20C for both rehydration and IEF. Rehydration time 12 hours.

Immobiline DryStrip Length pH range(s) 7cm* 3-10 3-10NL 4-7

11cm

3-10 4-7

13cm* 3-10 3-10NL 4-7

18cm

3-10 3-10NL 4-7

18cm

Narrow Intervals

24cm

3-10 3-10NL 4-7 3-7

24cm

Narrow Intervals

Step and voltage mode 1SH 2SH 3SH 4SH 5SH 6SH 7SH Total 1SH 2SH 3SH 4SH Total 1SH 2SH 3SH 4SH 5SH 6SH 7SH 8SH Total 1SH 2SH 3SH 4SH Total 1SH 2SH 3SH 4SH Total 1SH 2SH 3SH 4SH Total 1SH 2SH 3SH 4SH Total

Rehydration loading Step duration Voltage (V) (h:min) 20 12:00 100 1:00 500 1:00 1000 1:00 2000 1:00 4000 1:00 8000 4:00 21:00 20 12:00 500 1:00 1000 1:00 8000 1:50 15:50 20 12:00 100 2:00 500 1:00 1000 1:00 2000 2:00 4000 2:00 6000 2:00 8000 8:00 30:00 20 12:00 500 1:00 1000 1:00 8000 4:00 18:00 20 12:00 500 1:00 1000 1:00 8000 7:30 21:30 20 12:00 500 1:00 1000 1:00 8000 8:20 22:20 20 12:00 500 1:00 1000 1:00 8000 10:30 24:30

Volt-hours (kVh) 0.24 0.1 0.5 1 2 4 32 39.84 0.24 0.5 1 12.5 14.24 0.24 0.2 0.5 1 2 8 12 64 87.94 0.24 0.5 1 30.5 32.24 0.24 0.5 1 58.5 60.24 0.24 0.5 1 62.5 64.24 0.24 0.5 1 94.5 96

SH = Step and Hold *The 7 and 13cm protocol are currently the only protocols tested. All other protocols are suggested by the manufacturer.

10) Once the program is complete, turn off the power and remove your samples. 11) IPG strips can either be stored in a plastic Petri dish at -80C (gel side facing up) or directly following two equilibration steps for the further SDS-PAGE analysis. 12) The ceramic holders are cleaned with a special cleaning solution made by Amersham (see step 3). The gold plates on the IPGphors are cleaned with water only.

4. Second dimension Separation: SDS-Page

This protocol uses 140 x 150 x 1mm SDS-page (middle gel) as an example 1) Make a SDS polyacrylamide gel without the upper stack, leaving about 50mm of room from the top. 2) If your strips are stored at -80C, remove the IPG strips from the freezer and allow them to thaw for 10 to 20 minutes prior to equilibration of strips. Always use tweezers when handling strips. Tweezers should only touch the strips at the edges 3) Equilibration of IPG strips prior to SDS PAGE: First Equilibration with reductant: 15 min reduction with SDS-Equilibration buffer plus dithiothreitol (DTT). Second Equilibration with alkylating agent: 15 min alkylaion with SDSEquilibration buffer plus iodoacetamide (IAA). Both procedures are conducted while shaking the samples in solution. Make sure the procedure takes place while the IPG strips are facing upwards. 4) Prepare a protein standards marker: heat the protein standards at 90C for 3 minutes before applying it to a 50mm x 50mm piece of filter paper. For example, in Ref.3 for 255 x 196 x 1 mm gel, apply 200 to 1000 ng of each protein for Commasssie staining and about 10 to 50 ng of each protein for silver staining . (In fact 1ug of each protein of unstained protein marker is used to 140 x 150x 1 mm gel by Dr. Litchfield lab for SYPRO ruby staining gel) 5) Wash IPG strips with SDS PAGE running buffer (1x) before loading to the second-dimension gel in order to get rid of the free DTT and IAA. 6) Apply the equilibrated IPG strips to the second-dimension gel between the two glass plates. Make sure the IPG plastic backing is against ONE of the glass plates. Leave room on one side of the strip and place the filter paper with protein marker here. Tip: use pipette tips to push the strip down into two plates so it can to make contact with the gel.

7) Seal the IPG strip in place: Add about 1mL of agarose sealing mixture over the gel strip and filter paper to completely cover them. Allow 10 to 15 minutes to solidify. Tip: If Agarose sealing solution is already made and in a falcon tube, open the tube and place it face down in a glass beaker before putting the solution in the microwave. This will make for an easier (and cleaner) melting process 8) Run the gel: Generally, the higher the voltage, the faster your samples will run through the gel. However, at too high of a voltage melting as well as a pour visual resolution of your gel will occur. For Example: for a 12%, 14cm gel (middle-gel) it typically is run at 200V and 0.3A for 4.5hrs. 9) Stain the gel with Coomassie, Silver or SYPRO Ruby staining to determine protein levels or with ProQ Diamond staining to detect phosphorylated proteins.

Reagents 1% Bromophenol Blue Stock Solution: Solution Component Bromophenol blue Tris-base Deionized water (Milli Q)

Amount (10 mL) 100 mg 60 mg Up to 10 mL

Rehydration Buffer: Solution Component Amount (10 mL) Urea 4.2 g Thiourea 1.52 g CHAPS 0.4 g 1 M DTT* 500 L 100% IPG buffer (Ampholines)* 50 L 1% Bromophenol blue 200 L Deionized water (Milli Q) Up to 10 mL *If freezing Rehydration Buffer do not add DTT or IPG buffer until ready to use SDS Equilibration Buffer: Solution Component 1 M Tris, pH 8.8 Urea 100% Glycerol 10% SDS 1% Bromophenol Blue Deionized water (Milli Q)

Amount (200 mL) 10 mL 72.07 g 60 mL 40 mL 400 L to 200 mL

This is a stock solution. Prior to use DTT or iodoacetamide are added.

For SDS equilibration buffer with DTT-> add 100 mg DTT to 10 mL of buffer For SDS equilibration buffer with IAA-> add 250 mg IAA to 10 mL of buffer

12% SDS Polyacrylamide Gel: Solution Component Deionized water (Milli Q) 30% Acrylamide 1.5 M Tris, pH 8.8 10% SDS 10% APS TEMED

Amount (10 mL) 3.3 mL 4.0 mL 2.5 mL 100L 100L 4L

For different gel compositions please consult the source: Sambrook, J., and Russell, D.W., Molecular Cloning: A laboratory manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

Agarose Sealing Mixture: Solution Component 1X TAE Buffer Agarose 1% Bromophenol Blue
Microwave to boiling point to dissolve

Amount 50 mL 0.5 g 100 L

SDS PAGE Running Buffer (10x): Solution Component Glycine Tris base SDS ddH2O Amount (4L) 576g 121.1g 40g top up to 4L