Hendrik Poorter & Yvonne de Jong-Van Berkel
Hendrik Poorter IBG-2, Forschungszentrum Jülich
Yvonne de Jong-Van Berkel, Ecophysiology of Plants, Faculty of Science, Utrecht University
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.
- Sodium hydroxide 0.1 N
- Water bath
- Table centrifuge
- Centrifuge tubes
- 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).
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.