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. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput.
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 a 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. 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.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.
Sample Preparation for Western Blotting: Cell Lysis and Protein Extraction All the steps for protein extraction from cells or tissue (fresh or frozen) must be carried out at °C. The following is the composition of one common lysis buffer that is used to prepare protein samples. Cell lysis and protein extraction Historically, physical lysis was the method of choice for cell disruption and extraction of cellular contents; however, it often requires expensive, cumbersome equipment and involves protocols that can be difficult to repeat due to variability in the apparatus (such as loose-fitting compared with tight-fitting homogenization pestles).
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 . Reducing agents will be added into solution or buffer for protein extraction and purification to avoid the lost of activity of proteins or enzymes which is caused by oxidization.
Storage of proteins is important as the half-life of protein is commonly dependent on the storage temperature [ 4 ]. Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest.