Experiments for the determination of soluble and insoluble protein contents in plants
Experiments for the determination of soluble and insoluble protein contents in plants
Senescence is a biological phenomenon that develops in plants over a long period of evolution and natural selection, and it can be manifested at different levels such as cells, organs and the whole plant. During plant leaf senescence, chlorophyll content decreases, leaf color turns yellow, and protein content decreases. Also dysregulation of free radical metabolism and its accumulation in the body is one of the mechanisms of plant senescence. The main purpose of this experiment is to determine the changes of leaf proteins in the process of plant senescence, and to master the principle and method of determining soluble and insoluble proteins in plants.
Operation method
Experiments for the determination of soluble and insoluble protein contents in plants
Principle
After the plant material is ground in Tris-HCl buffer and centrifuged, the soluble proteins are dissolved in the supernatant and the insoluble proteins are present in the precipitated fraction. Hydrolyzing the precipitate with alkali results in an extract of insoluble protein. The protein extract reacts blue with Thomas Brilliant Blue G-250. Within a certain range, the protein content is proportional to the absorbance of the reaction solution at 595 nm, from which the protein content can be found.
Materials and Instruments
Plant Leaf Move 1. Preparation of a standard curve for bovine serum proteins For more product details, please visit Aladdin Scientific website.
Caulmers Brilliant Blue Solution HCl MgCl2 EDTA Dithiothreitol Bovine Serum Protein Standard Masterbatch
High-speed freezing centrifuge Spectrophotometer Constant temperature bath Mantle Test tubes Pipettes Earwash ball
(1) Take 6 test tubes, number them, and prepare the standard solution of bovine serum protein with the content of 0-100 μg in each tube according to the table below.
After adding the reagents in the table, shake well, take the tube 0 as the blank control, and measure the absorbance (A) value at 595nm. 
(2) Standard curve
Take the content of bovine serum protein as the horizontal coordinate and the absorbance value as the vertical coordinate to draw the standard curve.
2. Protein extraction
Weigh the leaves 0.3 ~ 0.5 g, cut in a frozen mortar with 3 ml of extract, add a small amount of quartz sand, quickly ground into a homogenate in an ice bath, homogenate poured into a centrifuge tube, and then 5 ml of extract (divided into two times) will be homogenized mortar washed into the centrifuge tube, and then centrifuged at 10000 r / min, 4 ℃ under the condition of 20 min, the supernatant is the extract of soluble proteins.
Add 3 ml of 1 mol-L-1NaOH solution to the precipitate obtained by centrifugation, and heat it in a constant temperature water bath at 90℃ for 20 min, then centrifuge it at 4000 r/min for 10 min at 4℃, and the supernatant is the insoluble protein extract.
3. Determination
Take 0.1 ml of each of the above two supernatants into two test tubes, add 0.9 ml of Tris buffer to each tube, add 1 ml of Tris buffer to the blank control tube, and then add 5 ml of Cauloblue staining solution to each tube, shake well, and then measure the absorbance (A) value at 595 nm.
V. Calculation of results
According to the measured absorbance value of the sample solution, find out the protein content from the standard curve, and calculate the soluble and insoluble protein content according to the following formula. 
