Protocol
Authors
Hendrik Poorter & Yvonne de Jong-Van Berkel
Author Affiliations
Hendrik Poorter IBG-2, Forschungszentrum Jülich
Yvonne de Jong-Van Berkel, Ecophysiology of Plants, Faculty of Science, Utrecht University
Overview
Protein can be extracted from fresh plant material after removing chlorophyll and other compounds such as amino acids using 80% acetone and methanol (chlorophyll extraction) or 80% ethanol (amino acid extraction). The extraction with sodium hydroxide gives a protein extract that can be used for determination of proteins using Bradford’s method.
Materials/Equipment
Reagents
- Sodium hydroxide 0.1 N
Equipment
- Water bath
- Table centrifuge
- Centrifuge tubes
Procedure
- Add 5 ml 0.1 N NaOH to the pellet after a macro chlorophyll extraction or 0.5 ml 0.1 N NaOH after a micro chlorophyll extraction (see Chlorophyll extraction and determination ).
- Mix thoroughly and put the test tubes in a waterbath of 70∘C.
- Centrifuge for 10 minutes at high speed in a table centrifuge (4500 rpm – 2694 g).
- Decant the supernatant in a volumetric flask of 25 ml or 10 ml.
- Do the same extraction two more times.
- Fill up the volumetric flask with 0.1 N NaOH.
- The extract is ready for determination using Bradford’s assay (reagents and kits commercially available, method not provided here).
Literature references
Davies, E.M., 1988. Protein assays: A review of common techniques. ABL 28-37.
Tal, M., Silberstein, A. & Nusser, E., 1980. Why does coomassie brilliant blue R interact differently with different proteins. J. Biol. Chem. 260: 9976-9980.
Sedmak, J.J. & Grossberg, S.E., 1977. A rapid sensitive and versatile assay for protein using coomassie brilliant blue G250. Anal. Biochem. 79: 544-552.