PNL Volume 11 1979 RESEARCH REPORTS 10
Hartmann, K. Institute of Genetics, Bonn, West Germany
Breeding for increased protein in field crops has been facilitated by the
availability of efficient screening methods. Esen (Anal. Biochem. 89:264-273.
1978) has recently published a simple determination method for quantitative
assay of protein which gives a linear response from 0.05 to 3-4 mg/ml of pro-
tein. Beside purified proteins like cytochrome C, Bovine serum albumin (BSA),
and egg albumin, Esen proposed the applicability of this method for protein
extractions of plant tissues. The method requires spotting of five-microliter
samples of the protein solutions in question on Whatman chromatography paper
No. 1. The sheet is stained in a 0.1% coomassie brilliant blue R-250 solution
[composed of 65 parts of distilled water, 25 parts of isopropyl alcohol, and
10 parts of acetic acid (v/v/v)] for 15 min. Then the paper is destained by
passing it through 3 glass trays, each containing distilled water. Following
drying, the stained protein spots are cut out in the form of disks with a
corkborer (19 mm diam). An unspotted disk serves as a blank. The dye-protein
PNL Volume 11
complex is eluted for 45 mm in test tubes containing 5 ml 0.1% sodium dodecyl
sulfate (SOS) solution. Then the solution is decanted into cuvettes and the
absorbance is read at 600 nm with a Beckman spectrophotometer against the eluate
of the blank set at 0.000 absorbance.
To determine the value of this assay in Pisum sativum seed protein, extrac-
tions, different amounts of seed flour of our initial line ('Dippes gelbe
Viktoria'J, were dissolved in a KCl-buffer (0.2 mol KC1, pH 6.85) to give an
estimated final concentration from 0.5 to 2.5 mg protein per ml. When the
extraction process lasts up to 48 hours, uniform and reproducible results were
obtained. Six replicates of each extraction were measured and compared with
a BSA regression line (Fig. 1) established by concentrations reaching from
0.5 to 2.5 mg protein per ml. These results are in good agreement with those
obtained by the Kjeldahl method. So far BSA seems to be a useful standard
tor estimating the seed protein values in Pisum until purified Pi sum seed
protein is available.
Fig. I. BSA-regression line established by concentrations reaching from
0.5 to 2.5 mg protein per ml.