This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes.
Share your feedback Abstract This protocol provides an efficient method for preparation of high-quality proteins from rice leaves and grains. The method involves phenol extraction to separate proteins from the non-protein components such as polysaccharides, lipids and phenolic compounds that are commonly enriched in plant tissues.
As the protocol is simple, universal, and most importantly compatible with silver staining, it has been applied to our routine protein extraction from rice and many other plant tissues and it even works fine in animal tissues for the requirement of electrophoretic separation. TNG67, an elite japonica type rice variety, has been a leading variety in Taiwan for more than 30 years since it been released in An aroma mutant, exhibits an agreeable taro-like flavor, was selected from the TNG67 mutation pool developed via sodium azide mutagenesis.
M 2-propanol Merck KGaA, catalog number: Grind the tissue to Extraction of proteins fine powder, using a mortar and pestle Figure 2B, Video 1. Add 5 ml extraction buffer and immediately grind the sample until foaming Figure 2C, Video 1.
Then add 5 ml extraction buffer and grind the sample until foaming Video 1. Transfer the homogenate into a sterilized 50 ml centrifuge tube Video 1. The remaining plant material sticking on mortar and pestle is rescued by carefully rinsing mortar and pestle with 5 ml extraction buffer, and poured into the tube and mixed well with the homogenate Video 1.
Centrifuge 20 min at 15, rpm in No. Filter the supernatant through 2 layers of Miracloth into another fresh tube Figure 2F. Extract the filtrate with an equal volume of buffer saturated phenol about 16 ml.
Mix well by inversion. Transfer the top layer phenol phase into another fresh tube Figure 2G and 2H. Preparation of protein sample. Leaves were ground and extracted by extraction buffer.
Protein sample was purified by phenol extraction. Extract the phenol phase with an equal volume of cold extraction buffer twice 1st 14 ml and 2nd 12 ml, respectively.
|DNA, RNA, and Protein Extraction: The Past and The Present||Purpose[ edit ] Protein purification is either preparative or analytical.|
Recover the top layer phenol phase. Add 3x volume of cooled precipitation buffer to the recovered phenol phase and mix well by inversion. Wash the pellet with 1. Recover the pellet by centrifugation at 8, rpm for 5 min.
Dry the pellet by SpeedVac for about min less than 5 min and resuspend in an appropriate volume of lysis buffer. The obtained protein samples can be used for proteomic analysis Lin et al.
Proteomic analysis and Western blot analysis. Proteomic analysis of leaf proteins from various rice varieties. Preparation of the protein samples from rice grains Freeze 0.
Place the frozen tissue in a mortar containing little seasand and liquid nitrogen. Grind the tissue to a powder with the mortar and pestle.
Grind sample for 2 min or to a homogenate and then transfer into a sterilized 50 ml centrifuge tube. Transfer the supernatant into another fresh tube.
Add 3x volume of cold 0. Dry the pellet by the SpeedVac system for about min less than 5 min and resuspend in an appropriate volume of lysis buffer. The obtained protein samples can be used for proteomic analysis or Western blot. Representative data Video 1. Protein extraction Notes In this protocol, the critical steps which affect the yield and intactness of protein samples are as follows: The lyophilized sample is not recommended, because some dehydrated proteins might be difficult to dissolve in the extraction buffer.
The complete homogenization can be achieved by grinding the samples in extraction buffer see Video 1. Do not dry the pellet too long in the step B9.Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods.
The methodical comparison of protein isolation methods is the first critical step for proteomic studies.
MicroRotofor lysis kits provide cell lysis and protein extraction protocols that are tailored to the specific needs of different sample sources. ReadyPrep™ Mini Grinders ReadyPrep mini grinders are used in sample extraction protocols to grind small biological samples .
Protocols for the extraction of proteins have also been steadily improving over the last few decades,, and it is possible to take advantage of the similarities between these techniques and metabolomics extraction techniques to extend current methods. Key words: rice proteins, extraction, denaturation, and hydrophobicity.
Introduction T HERE HAS BEEN INCREASED USE OF RICE PRODUCTS AS IN-gredients in gels, puddings, ice creams, and baby formu- Extraction, denaturation and hydrophobic Properties of Rice Flour Proteins.
Protein Extraction Each group should select a source of protein for their study. The class might like to compare measured protein content in some plant-based foods to the amount reported on packaging.
Membrane proteins are embedded in the lipid bilayer, held in place by one or more domains spanning the hydrophobic core. In addition, peripheral proteins bind the inner or outer surface of the bilayer through interactions with integral membrane proteins or with polar lipid head groups.
Cell lysis and protein extraction Historically.