四、www.biomart.cn/runwell/index.htm
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
up to 100 ml with dH20. pH 7.4. Then 0.2 um filter.
1. Freeze tissue in liquid nitrogen.
2. Rinse in PBS then mince.
3. Add 1 ml Camiolo extraction buffer per 100 mg of tissue.
4. Homogenize for 1 minute at 4\'C.
5. Spin at 3,000. rpm/15 minutes/4\'C.
6. Remove supernatant and save in another tube.
7. If necessary, dialize the supernatant against PBS with
50mM/L Tris-HCl pH 7.4.

五、www.runwelltac.com
植物材料：水稻苗，葉鞘，根（ynibcas）
1、200 毫克樣品置于冰上磨碎
2、加lysis buffer，離心，10000rpm，4度，5min 取上清
3、重復離心5min
lysis buffer：urea np-40 ampholine 2-me pvp-40

六、
蛋白質樣品制備（sigma）
秧苗蛋白質樣品的提取按Davermal 等（1986）的方法進行。
100mg材料剪碎后加入10mgPVP-40（聚乙烯吡咯烷酮）及少量石英砂，用液氮研磨成粉，加入1.5 ml 10% 三
氯yi酸（丙酮配制，含10mM 即0.07%β-巰基乙醇），混勻，-20℃沉淀1 小時，4℃，15000 r/min離心15
min，棄上清，沉淀復溶于1.5ml冷丙酮（含10 mMβ-巰基乙醇），再于-20℃沉淀1 小時，同上離心棄上清，
（有必要再用80％丙酮（含10 mMβ-巰基乙醇所得沉淀低溫冷凍真空抽干。
按每mg干粉加入20μl（可調） UKS液[9.5 M尿素，5mM 碳酸鉀，1.25%SDS，0.5%DTT（二硫蘇糖醇），
2% Ampholine （Amersham Pharmacia Biotech Inc，pH3.5-10），6% Triton X-100]，37℃溫育30min，期間攪
動幾次，28度 （溫度低，高濃度的尿素會讓溶液結冰）16000 r/min離心15 min，離心力越大時間長一點
越好！上清即可上樣電泳。或者-70 度保存

七、
植物根中蛋白質的抽取（phenol）
（1） sample, 液氮研磨
（2） 裝1.5 ml centrifuge 用tube
（3） 加 1M KH2PO4+K2HPO4 700 ul
（4） 12000 rpm, 4度, 10-15minite
（5） 取上層液，蛋白質就在里面

八、
SDS extraction followed by acetone precipitation – simple extraction protocol that does not require phenol.
Recommended start protocol for whole tissue extractions.（hgp）
1. Grind 1 g of fresh tissue to a powder with liquid nitrogen in a mortar and pestle.
2. Add 5 mL of extraction media （0.175 M Tris-HCl, pH 8.8, 5% SDS, 15% glycerol, 0.3 M DTT） directly to
mortar and continue grinding for an additional 30 sec.
3. Filter homogenate through two layers of miracloth into a 50 mL Falcon tube at room temperature.
4. Immediay add 4 volumes of ice cold 100% acetone to filtered homogenate, mix by vortexing and place at -20
C for at least one hour to precipitate proteins.
5. Centrifuge at 5000 g for 15 min to collect precipitated protein, decant supernatant.
6. Gently blot residual acetone from container with Kimwipe and then wash pellet in 15-20 mL of cold 80%
acetone. Be sure to thoroughly break-up pellet by pipetting, vortexing or sonication.
7. Repeat steps 5 and 6.
8. Collect final protein precipitate by centrifugation at 5000 g for 15 min and dry pellet by inverting on Kimwipe
for 15 min at 37 C.
9. Resuspend final pellet in 0.5-1 mL of IEF extraction solution （8 M urea, 2 M thiourea, 2% CHAPS, 2% Triton
X-100, 50 mM DTT, 0.2% pH 3-10 ampholytes） by pipetting and vortexing at 25-30 C. Incubate sample for 1 h at
room temperature with agitation. Do not heat sample under any circumstances as this will lead to carbamylation of
proteins.
10. Centrifuge for 10 min at 12000 g and use supernatant to rehydrate IPG strips.
11. If protein quantitation is necessary, precipitate protein sample with TCA or acetone prior to performing
Bradford or Lowry assay as detergents and reducing agents interfere with these assays.
Phenol extraction followed by methanolic ammonium acetate precipitation – an effective protocol for sample
preparation from protein-poor, recalcitrant tissues such as plants （see Hurkman and Tanaka, 1986, Plant
Physiology 81:802-80

九、
材料：細菌蛋白（puc18）
用甲醇提取的，凍干后用緩沖液溶解的。樣品緩沖液是一般的。其中含又2%的SDS，20mmol 的2-巰基乙
醇。

十、
線粒體蛋白的提取 （bioon）
Isolation for Mitochondria
Modification by Bioon
Materials and reagents:
homogenizing buffer:
100 mM mannitol
10 mM Tris-HCl buffer （pH 7.5）
5 mM MgCl2
關 于 蛋 白 質

1 mM EGTA
1 mM DTT
leupeptin （0.1 ug/ml）
0.1M Na2CO3
Methods:
- 10*6 Cells were washed with ice-cold PBS and lysed by homogenizing in 1 ml buffer （ice-cold） containing 100
mM mannitol, 10 mM Tris, 5 mM MgCl2, 1 mM EGTA, 1 mM DTT, leupeptin （0.1 ug/ml）
- Subjected to Polytron homogenization for three-four bursts of 3-10 s each at a setting of 6.5.
- Intact cells and nuclei were separated by centrifugation at 120 g for 5 min at 4℃
- Supernatants were centrifuged at 10,000 g for 10 min to collect the heavy （mitochondrial） membrane pellet.
- Cytoplasmic fractions were obtained by centrifuging supernatants at 100,000 g for 30 min.
- Resuspended pellet to 0.25mg/ml in fresh preparation of 0.1M Na2CO3 （pH 11.5）
- Incubated on ice for 30 min.
- Ultracentrifugation at 100000g for 1h at 4℃ to precipitate the mitochondria membrane protein. And the
supernatants are mitochondrial matrix. 0.5mg of proteins in mitochondria can get 100ug of proteins （the
alkali-resistant fractions）
Ref.: PNAS, 2002，99：12825–12830
本方法只適用于提大鼠細胞線粒體蛋白，而不適用于線粒體功能檢測