SIMPLE PROTEIN ASSAY FOR PEA

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

RLSHARCH REPORTS

PNL Volume 11

1979

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.



	PNL Volume 11 1979 RESEARCH REPORTS 10

	SIMPLE PROTEIN ASSAY FOR PEA 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