Immobilized pH Gradient (IPG)—1st Dimension Electrophoresis
Sample Resolubilization
Protein samples or whole cell lysates from TCA precipitation are rehydrated in lysis/rehydration buffer at a concentration of 7 mg/ml for staining with Coomassie. Samples obtained from TCA precipitation may need pH adjustment with ammonia hydroxide. Whole cell pellets are estimated to be ca. 7% protein. Membrane proteins are estimated at 10% of the total protein. The samples are incubated with lysis/rehydration buffer with vortex mixing at 5 min intervals for 20 minutes at room temperature or resuspended by sonication. Insoluble debris is removed by centrifugation at 16,000 rpm for 15 minutes. Standard format for the second dimension analysis is a 20 cm x 20 cm and 1.5 mm thick gel, requiring an 18 cm IPG strip (Amersham Pharmacia Biotech). Typically 1-3 mgs of a complex protein mixture is needed per gel for staining by Coomassie Blue. Silver stain and fluorescence staining are more sensitive and are available for low-level samples. Each gel requires 100-300 mg of sample.
Rehydration of IPG strips with sample
An aliquot of sample in lysis/rehydration buffer (20 ml per cm of IPG strip, i.e. 360 ml for an 18 cm IPG gel strip) is placed in a rehydration tray and an IPG strip is placed gel side down on the sample. The gel is covered with ca. 2 ml paraffin/mineral oil and left to rehydrate for 18-20 hour.
Multiphor II Focusing
Following rehydration, the IPG strips are rinsed with Milli-Q water, placed on a strip alignment sheet. The alignment sheet is placed in a Multiphor II unit (Amersham Pharmacia Biotech) cooled to 20°C. Isoelectric focusing begins at low voltage (150 V for 1 hr). The voltage is gradually increased to 6000 V over a 5 hr period and then run for approximately 120 kVhr for 18 cm strips (<100 kVhr for 11 cm strips and <75 kVhr for 7 cm strips). After focusing, the IPG strips are removed and rinsed with MilliQ water, then equilibrated as described below. Alternatively, IPG strips may be frozen at –80°C.
Equilibration of IPG Sample Strips
Following focusing, the IPG strips are rinsed with MilliQ-Q water and equilibrated with ca. 10 ml TBP buffer for 20 min to reduce protein disulfide bonds. Sulfhydryl groups are alkylated with iodoacetamide (IAA) for 20 min. Reduction and alkylation are done at room temperature.
Solutions
Lysis/Rehydration buffer
Solubilization of protein samples for rehydration of IPG strips (Amersham Pharmacia Biotech) is performed in the following buffer: 7M urea, 2M thiourea, 0.002% bromophenol blue (as tracking dye), zwitterionic and/or non-ionic detergent (e.g. 0.8% CHAPS, 1% Triton X-100, or a variety of other detergents), 2 mM tributyl phosphine (TBP) and 0.5% IPG Buffer (Amersham Pharmacia Biotech). Protease and phosphatase inhibitors are included as necessary.
Stock solubilization/rehydration Buffer (incomplete)
7 M urea
2 M thiourea
0.8% CHAPS
1% Triton X-100
(additional/alternative non-ionic or zwitterionic detergents as desired; e.g. another common detergent combination used by the MPC is 0.8% CHAPS, 1% Triton-X100 and 1% ASB-14)
0.002% Bromophenol Blue
Prepare in batches/aliquots of 5ml and /or 1ml and store frozen at –80°C.
Stock 100mM tributyl phosphine (TBP):
(Make up fresh every 2 weeks or less)
4.75 ml isopropanol
0.249 ml tributyl phosphine
Stock solubilization/rehydration Buffer (complete) 10ml
make fresh
10 ml of Stock solubilization/rehydration buffer (incomplete)
10 ml of IPG Buffer (select a pH range to match that of the IPG gel strip, Amersham Pharmacia Biotech), 0.5% final
10 ml of stock TBP; 2 mM final
Protease and phosphatase inhibitors as desired
Suggested Protease Inhibitors
protease inhibitor cocktail, e.g. Complete, Mini (Roche); 1 tablet per 10 ml buffer
Serine protease inhibitor, 1mM phenymethylsulfonylfluoride (PMSF)
Phosphatase inhibitors, 0.2 mM Na vanadate and 5 mM NaF
Stock Equilibration buffer
50 mM Tris-HCl, pH 8.8
6M urea
20% glycerol
2% SDS
Store at –20°C in 50 ml aliquots
TBP Buffer: 50 ml stock equilibration buffer plus 1.25 ml of 100 mM TBP solution; 2.5 mM TBP final
IAA Buffer: 50 ml stock equilibration buffer plus 1.25 g iodoacetamide (IAA), 135 mM IAA
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Protocols
The Michigan Proteome Consortium follows a standard set of protocols listed below:
Whole cell lysate
Membrane proteins
IPG 1st dimension
SDS page
Colloidal coomassie stain
Fluorescence stain
Silver stain
In-gel digestion
References and suggested reading
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