Method for extracting plant protein by high speed refrigerating centrifuge
First, plant tissue protein extraction method Fifth, protein sample preparation More centrifuges, high-speed refrigerated centrifuge information registration: Lower Blood Sugar Plant Extract Lower Blood Sugar Plant Extract,Inonotus Obliquus Extract,Chaga Extract Powder,Chaga Mushroom Extract Powder Polysaccharide Fufeng Sinuote Biotechnology Co.,Ltd. , https://www.ffsinuoteplant.com
　　
1. According to the weight of the sample (3.5 g of the extract is added to 1 g of the sample, it can be added according to the material), and the extract is prepared on ice.
　　
2. The sample was placed in a mortar and ground with liquid nitrogen. After grinding, it was added to the extract and allowed to stand on ice (3-4 hours).
　　
3. Centrifuge with centrifuge 8000 rpm 40 min 4 ° C or 11100 rpm 20 min 4 ° C
　　
4. The supernatant is extracted and the sample preparation is completed.
　　
Protein extract: 300ml
　　
1, 1Mtris-HCl (PH8) 45ml
　　
2, glycerin (Glycerol) 75ml
　　
3, polyvinylpyrrolidone (Polyvinylpolypyrrordone) 6g
　　
This method is for SDS-PAGE, vertical plate electrophoresis!
　　
Second, plant tissue protein extraction process
　　
Chloroacetic acid-acetone precipitation
　　
1. Grinding blades in liquid nitrogen
　　
2. Add 3 times the sample volume to the extract at -20 ° C overnight, then centrifuge (4 ° C for more than 8000 rpm for 1 hour) to discard the supernatant.
　　
3. Add an equal volume of ice bath acetone (containing 0.07% β-mercaptoethanol), shake it and centrifuge (4 ° C for more than 8000 rpm for 1 hour), then dry the precipitate in vacuo and set aside.
　　
4. Add the lysate before loading, leave it at room temperature for 30 minutes, dissolve the protein in the lysate, and then centrifuge (15 ° C 8000 rpm for 1 hour or longer without precipitation as the standard), can be temporarily stored at 4 ° C use.
　　
5. The protein was quantified by the Brandford method and then dispensed at -80 °C for later use.
　　
Drug: Extract: Acetone containing 10% TCA and 0.07% β-mercaptoethanol. Lysate: 2.7 g of urea 0.2 g of CHAPS was dissolved in 3 ml of sterilized deionized water (final volume of 5 ml), and 1 M of DTT 65 ul/ml was added before use.
　　
This method is aimed at two-dimensional electrophoresis, with less impurities and a small ion concentration! Of course, one-way electrophoresis is also applicable, but the strip of electrophoresis will be reduced!
　　
Third, tissue: intestinal mucosa
　　
Objective: WESTERNBLOT detects the expression of apoptosis-related proteins
　　
Steps to extract proteins using TRIPURE:
　　
Add isopropyl alcohol to the protein-containing supernatant: (1.5ml per 1ml of TRIPURE)
　　
Invert and mix, set at room temperature for 10min
　　
Centrifugation: 12000g, 10min, 4 degrees, discard the supernatant
　　
Add 0.3M guanidine hydrochloride / 95% ethanol: (2ml per 1ml TRIPURE)
　　
Oscillating, set at room temperature for 20min
　　
Centrifugation: 7500g, 5min, 4 degrees, discard the supernatant
　　
Repeat 0.3M guanidine hydrochloride / 95% ethanol step 2 times
　　
Add 100% ethanol to the precipitate and add 2ml
　　
Shake well and mix well, set at room temperature for 20min
　　
Centrifugation: 7500g, 5min, 4 degrees, discard the supernatant and dry the precipitate
　　
1% SDS dissolved precipitate
　　
Centrifugation: 10000g, 10min, 4 degrees
　　
Take a clear -20 degree save (or can be used directly in WESTERNBLOT)
　　
There is a problem: the precipitate does not dissolve after adding 1% SDS, or a large piece, after the 4 degree centrifugation, there is more white sedimentation, SDS crystallization? The concentration was measured and the content was only about 1 mg/ml.
　　
Solution: Lift the protein kit, the other tissue size is moderate, to be broken, immediately add 2XBUFFER, then cook for 5-10 minutes, the effect is very good.
　　
Fourth, plant materials: rice seedlings, leaf sheaths, roots
　　
1, 200 mg sample is ground on ice
　　
2, add lysisbuffer, centrifuge, 10000 rpm, 4 degrees, 5 minutes to take the supernatant
　　
3, repeated centrifugation for 5min
　　
Detectbuffer:ureanp-40ampholine2-mepvp-40
　　
The extraction of the seedling protein sample was carried out in accordance with the method of Davermal et al. (1986).
　　
After cutting 100mg of material, add 10mg of PVP-40 (polyvinylpyrrolidone) and a small amount of quartz sand, grind it into powder with liquid nitrogen, add 1.5ml of 10% trichloroacetic acid (formed with acetone, containing 10mM or 0.07% β-mercaptoethanol), and mix. Precipitate at -20 °C for 1 hour, centrifuge at 15000 r/min for 15 min at 4 ° C, discard the supernatant, dissolve the pellet in 1.5 ml of cold acetone (containing 10 mM β-mercaptoethanol), and precipitate at -20 ° C for 1 hour. Clear, (it is necessary to use 80% acetone (containing 10 mM β-mercaptoethanol to obtain a precipitate and freeze it under vacuum).
　　
20 μl (adjustable) UKS solution [9.5 M urea, 5 mM potassium carbonate, 1.25% SDS, 0.5% DTT (dithiothreitol), 2% Ampholine (Amersham Pharmacia Biotech Inc, pH 3.5-10), 6 mg per mg dry powder %TritonX-100], incubate at 37 °C for 30 min, stir a few times, 28 degrees (low temperature, high concentration of urea will make the solution freeze) 16000r / min centrifugation for 15min, the greater the centrifugal force, the longer the better! The supernatant can be loaded with electrophoresis. Or -70 degrees to save
　　
6. Extraction of protein from plant roots
　　
(1) sample, liquid nitrogen grinding
　　
(2) with 1.5mlcentrifuge with tube
　　
(3) plus 1MKH2PO4K2HPO4700ul
　　
(4) 12000 rpm, 4 degrees, 10-15 minite
　　
(5) Take the upper layer of liquid, the protein is inside