Pioneer Hi-Bred International

Reliability of the protein extraction procedure used to prepare seedling samples for two-dimensional electrophoresis

--J. W. Higginbotham* and J. S. C. Smith

*Also affiliated with Cold Spring Harbor Laboratory, NY. As part of the collaboration with Cold Spring Harbor Lab. and Pioneer, two-dimensional electrophoretic profiles of corn seedling proteins are being computer analyzed with the software developed by Garrels (J. Biol. Chem. 254:7961, 1979). Because computer analysis discerns quantitative differences among proteins with unprecedented precision, it is extremely important to be aware of those factors which may cause quantitative differences in proteins among gels or distortion of the profile and to minimize the differences or distortion due to laboratory manipulation. With these objectives, three components of the corn seedling protein extraction procedure used at Pioneer were tested.

Protein extraction followed Damerval (Electrophoresis 7:52, 1986) with a few exceptions. Embryos were homogenized with a Brinkman polytron directly in cold acetone, the TCA precipitation step being omitted. Protamine sulfate was added to the extraction solution, but ampholytes were not included.

The TCA-precipitable radioactivity in the acetone supernatant was assayed to determine how much lipophilic protein was being lost in the initial extraction. The TCA-precipitable radioactivity of the sample is routinely assayed. Repeated assays revealed 1000-fold less labeled protein in the acetone than in the sample. Samples often contained 100,000cpm/ul of TCA-precipitable radioactivity. Proportional amounts of acetone supernatants contain an average of 100cpm.

Protamine sulfate (PS) is reported to precipitate nucleic acids (Mayer et al., Plant Cell Rep. 6:77, 1987), but its effectiveness in a high molar urea solution was unknown. Samples with and without PS in the extraction solution were electrophoresed at Johnston, and the gels silver stained to determine the effectiveness of this chemical in removing nucleic acids. In those without PS, numerous horizontal gray streaks extended across the gels. Samples prepared with PS had a much clearer background due to the almost complete absence of gray streaks.

Lastly, the effectiveness of the procedure in preventing degradation due to proteinase activity was investigated in three ways. Two samples were divided into two aliquots each. One aliquot of each sample was incubated at 37 C for 3 hrs prior to electrophoresis (at Johnston). The other aliquots were warmed to 37 C immediately prior to electrophoresis. Gels were subsequently silver-stained to determine if high-molecular weight proteins had been preferentially lost and if there was visible degradation of the protein profiles in the aliquots that had been kept at 37 C for 3 hrs.

Proteinase activity was assayed using Azocoll dye-bound collagen as the substrate. Proteinase K and trypsin were used as positive controls. One assay was performed using protein extraction solution instead of buffer in the reaction mixtures. Other assays were performed using .1M sodium phosphate buffer. For the latter assay, embryos were homogenized in cold acetone, the pellet dried under vacuum, and resuspended in .1M phosphate buffer. All reaction mixtures and blanks were read at 520nm.

The extraction procedure appeared to be effective in preventing degradation due to proteinase activity. The pairs of silver stained protein profiles were essentially identical with no preferential loss of high molecular weight proteins or visible degradation of the protein profiles.

No proteinase activity in the samples was detected using Azocoll substrate even when the reaction was allowed to proceed 30 min at 37 C. When protein extraction solution was used instead of phosphate buffer, no proteinase activity was detected in any of the positive controls either.

In summary, the results presented here indicate that the protein extraction procedure used routinely in our lab is a reliable, reproducible method for extracting corn seedling proteins. The variability generated due to quantitative loss of protein into the acetone supernatant during protein extraction appears to be very minimal. The removal of nucleic acids by protamine sulfate precludes distortion of the profile due to their presence. There appears to be little if any variability generated by the action of proteinases in situ. Not only does the extraction solution prevent proteinase activity, the acetone extraction appears to be an effective inhibitor of activity, too. Furthermore, samples appear to be stable at 37 C for at least 3 hrs. Samples can be stored frozen and warmed for reuse many times without risk of degradation.

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors

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