1. Protein extraction method of plant tissue (summer)
1. According to the weight of the sample (add 3.5ml of extraction solution to 1g of sample, which can be added according to different materials), prepare the extraction solution on ice
2. Place the sample in a mortar and grind it with liquid nitrogen. After grinding, add it to the extract and let it stand on ice (3-4 hours).
3. Centrifuge with a centrifuge at 8000 rpm, 40 min 4 ° C or 11100 rpm, 20 min 4 ° C
4. Extract the supernatant night and the sample preparation is completed.
Protein extract: 300ml
1. 1Mtris-HCl (PH8) 45ml
2. Glycerol 75ml
3.Polyvinylpolypyrrordone 6g
This method is for SDS-PAGE, vertical plate electrophoresis!
2. Protein extraction method of plant tissue (summer)
Trichloroacetic acid-acetone precipitation method
1. Grind the blade in liquid nitrogen
2. Add 3 times the volume of the extract to the sample overnight at -20 ° C, then centrifuge (at 8000rpm for more than 1 hour at 4 ° C) and discard the supernatant.
3. Add an equal volume of ice bath acetone (containing 0.07% β-mercaptoethanol), shake well and centrifuge (at 8000 rpm for more than 1 hour at 4 ° C), then vacuum
Dry the precipitate and set aside.
4. Add lysate before loading, let it stand at room temperature for 30 minutes to make the protein fully dissolved in the lysate, and then centrifuge (15 ℃ at 8000rpm for 1 hour)
If there is no precipitation for a period of time or longer), it can be temporarily stored at 4 ℃ for use.
5. Use the Brandford method to quantify the protein, and then put it into -80 ℃ for use.
drug:
Extract: Acetone with 10% TCA and 0.07% β-mercaptoethanol
Lysis solution: 2.7g urea 0.2g CHAPS dissolved in 3ml sterilized deionized water (final volume is 5ml), add 1M before use
DTT65ul / ml.
This method is aimed at two-dimensional electrophoresis, with less impurities and low ion concentration! Of course, one-way electrophoresis is also applicable, but the band of electrophoresis will be reduced!
three,
Tissue: Intestinal mucosa (newinbio)
Purpose: WESTERN BLOT to detect the expression of apoptosis-related proteins
Apply TRIPURE protein extraction steps:
Add isopropanol to the protein-containing supernatant: (1.5ml per 1ml TRIPURE dosage)
Invert and mix well, set at room temperature for 10min
Centrifuge: 12000 g, 10min, 4 degrees, discard supernatant
Add 0.3M guanidine hydrochloride / 95% ethanol: (2ml per 1ml TRIPURE dosage)
Shake, set at room temperature for 20min
Centrifuge: 7500g, 5 min, 4 degrees, discard supernatant
Repeat the step of 0.3M guanidine hydrochloride / 95% ethanol 2 times
Add 2ml of 100% ethanol to the precipitate
Shake well and mix well. Leave at room temperature for 20 min
Centrifugation: 7500g, 5min, 4 degrees, discard the supernatant and blow dry the sediment
1% SDS dissolved precipitation
Centrifuge: 10000g, 10min, 4 degrees
Take the supernatant and save at -20 degrees (or can be used directly in WESTERN BLOT)
Existing problems: After adding 1% SDS, the precipitate does not dissolve, or it is still a big piece. After centrifugation at 4 degrees, there is more white precipitation, and SDS crystallizes?
The concentration is only about 1mg / ml.
Solution: The protein extraction kit, in addition, the tissue size is moderate, to be broken, immediately add 2X BUFFER, and cook for 5-10 minutes, the effect is very good.
Four, lysis solution: (yog)
Protein extraction buffer (Camiolo buffer):
100 ml = (0.075M Potassium Acetate) 0.736g
(0.3M) NaCl 1.753g
(0.1M) L-arginine basic salt 1.742g
(0.01M) EDTA-HCl 0.292g
(0.25%) Triton X-100 250. ul
5. Plant materials: rice seedlings, leaf sheaths, roots (ynibcas)
1. 200 mg sample is ground on ice
2. Add lysis buffer, centrifuge, 10000rpm, 4 degrees, take the supernatant for 5min
3. Repeat centrifugation for 5min
lysis buffer: urea np-40 ampholine 2-me pvp-40
six,
Protein sample preparation (sigma)
Seedling protein samples were extracted according to 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 1.5 ml of 10%
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