Whole Cell Lysate Preparation
Sample concentration
Standard format for 2-D analysis is a 20 cm x 20 cm and 1.5 mm thick gel, requiring an 18 cm immobilized pH gradient (IPG) strip (Amersham Pharmacia Biotech). Typically 1-3 mg of a complex protein mixture is needed per 2-D gel for staining by Coomassie Blue. Silver stain and fluorescence staining are more sensitive and are available for low-level samples. Each gel requires 100-300µg of sample.
Protein estimations are based on the following generalizations. Whole cell pellets are estimated to be ca. 7% protein. Membrane proteins are estimated at 10% of the total protein or 0.7% of the whole cell pellet. Wet weight of whole cell pellet is estimated from packed cell volume. Assume a density of 1 g/ml.
Preparation
Harvest cells and wash thoroughly with phosphate buffered saline (PBS) buffer. Store cell pellets at -80oC. Cells are resuspended with 100 µl Tris/SDS/PI lysis buffer per 50 mg cell wet weight (ca. 350 µg protein). The samples are kept on ice and sonicated on low power setting with five 3-4 second bursts with 1 min between bursts. Add 20 µl of DNase/RNase solution per 400 µl of lysate. Incubate on ice for 15 min. Precipitate proteins by TCA precipitation (below).
Solutions
PBS Buffer:
10 mM potassium phosphate, pH 7.4
150 mM NaCl
Tris/SDS/PI Lysis Buffer:
50 mM Tris/HCl, pH 8.0
2% SDS
Protease and phosphatase Inhibitors as desired
DNase/RNase Stock Solution:
DNase I, 1 mg/ml
RNase, 0.25 mg/ml
500 mM Tris/HCl, pH 7.0
50 mM MgCl2
Suggested Protease and Phosphatase Inhibitors
protease inhibitor cocktail, 1 tablet Complete, Mini (Roche Molecular Biochemicals) per 10 ml lysis buffer
serine protease inhibitor: 1mM PMSF
phosphatase inhibitors (0.2 mM Na vanadate and 5 mM NaF)
TCA Precipitation
Trichloroacetic acid (TCA) is added to the sample to a 17% final concentration. The sample is mixed thoroughly and allowed to precipitate at -20oC for 2 hr to overnight. Centrifuge at 14,000 rpm and 4oC for 15 min. Remove supernatant and suspend the precipitated protein in HPLC grade acetone. Place at -20 oC for 1 hr and centrifuge at 14,000 rpm and 4oC for 15 min. Repeat the acetone washing step. Suspend the precipitate in ca. 50 µl water and lyophilize.
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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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