An optimized procedure for protein extraction and 2-D electrophoresis
of maize embryo proteins
--Guy Farish and William F. Sheridan
In our laboratory, we have optimized procedures for extracting maize embryo proteins, and their subsequent analysis using 2-D electrophoresis.
Our protein extraction is a modification of the methods of Laemmli (1970) and O'Farrell (1975). Isolated embryos were crushed in liquid nitrogen and boiled for 5 minutes in 300µl of a buffer containing 62.5mM Tris-HCl pH 6.8, 5% SDS, 5% b-mercaptoethanol, and 10% glcerol. The sample was then spun for 3 minutes in a microfuge and the supematant recovered. This extract was precipitated with 9 volumes of acetone at -20C for 2 hours, the precipitate spun down and dried. The precipitate was resuspended in 20 ml of lysis buffer containing 20mM Tris-Hcl pH 7.6, 5mM MgCl2, 1% ml of 2 mg/ml Dnase I and 1 ml of 1mg/ml Rnase. After nuclease treatment, 25 mg of urea and 40 ml of sample buffer (2% Nonidet P-40, 2% ampholytes, 5% BME, and 9.5M urea) were added to the sample. The samples were then stored at -80C.
Samples were assayed for protein content by taking a 10µl aliquot and precipitating it with 9 volumes of acetone at -20 C. This precipitate was redissolved in 0.1N NaOH, a standard Lowry assay performed and the samples read in a spectrophotometer at 750nm. This avoided the interference problems that most protein assays have with samples containing high concentrations of urea or detergents. A sample from a single mature embryo contained on average 14µg/µl protein.
Isoelectric focusing was performed using 2.0mm internal diameter glass tubes and gels were poured to 15cm in length. This large size allowed good resolution of 50-100µg of total protein. We used a 3:1 ratio of ampholytes (.75ml pH 5-7, .25ml pH 3-10) using Bio-Rad Biolyte ampholytes. Samples were focused for 20 hours at 500 volts plus 4 hours at 800 volts for a total of 13,200 volt/hours.
Second dimension separation was performed on uniform 12.5% polyacrylamide gels at pH 8.8 which are 16 x 18cm x 1.5mm in size. Gels were run 4-5 hours at 75mA/gel using constant current.
These conditions have given us excellent resolution and good reproducibility. Gels are commonly silver stained or blotted onto nitrocellulose.
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