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蛋白質(zhì)提取方法

2010-03-23 [5835]

蛋白質(zhì)提取方法-------列舉10種方法


一、
植物組織蛋白質(zhì)提取方法(summer)
1、根據(jù)樣品重量(1g樣品加入3.5ml提取液,可根據(jù)材料不同適當(dāng)加入),準(zhǔn)備提取液放在冰上。
2、把樣品放在研缽中用液氮研磨,研磨后加入提取液中在冰上靜置(3-4 小時(shí))。
3、用離心機(jī)離心8000rpm40min4℃或11100rpm20min4℃
4、提取上清夜,樣品制備完成。
蛋白質(zhì)提取液:300ml
1、1Mtris-HCl(PH8) 45ml
2、甘油(Glycerol)75ml
3、聚乙烯吡咯烷酮(Polyvinylpolypyrrordone)6g
這種方法針對(duì)SDS-PAGE,垂直板電泳!

二、
植物組織蛋白質(zhì)提取方法 (summer)
三氯醋酸—丙酮沉淀法
1、在液氮中研磨葉片
2、加入樣品體積3倍的提取液在-20℃的條件下過夜,然后離心(4℃8000rpm以上1小時(shí))棄上清。
3、加入等體積的冰浴丙酮(含0.07%的β-巰基乙醇),搖勻后離心(4℃8000rpm以上1 小時(shí)),然后真空
干燥沉淀,備用。
4、上樣前加入裂解液,室溫放置30 分鐘,使蛋白充分溶于裂解液中,然后離心(15℃8000rpm以上1小
時(shí)或更長(zhǎng)時(shí)間以沒有沉淀為標(biāo)準(zhǔn)),可臨時(shí)保存在4℃待用。
5、用Brandford法定量蛋白,然后可分裝放入-80℃?zhèn)溆谩?br />藥品:
提取液:含10%TCA 和0.07%的β-巰基乙醇的丙酮
裂解液:2.7g 尿素0.2gCHAPS 溶于3ml 滅菌的去離子水中(終體積為5ml),使用前再加入1M 的
DTT65ul/ml。
這種方法針對(duì)雙向電泳,雜質(zhì)少,離子濃度小的特點(diǎn)!當(dāng)然單向電泳也同樣適用,只是電泳的條帶會(huì)減少!

三、
組織:腸黏膜 (newinbio)
目的:WESTERN BLOT檢測(cè)凋亡相關(guān)蛋白的表達(dá)
應(yīng)用TRIPURE 提取蛋白質(zhì)步驟:
含蛋白質(zhì)上清液中加入異丙醇:(1.5ml每1mlTRIPURE用量)
倒轉(zhuǎn)混勻,置室溫10min
離心:12000 g,10min,4度,棄上清
加入0.3M鹽酸胍/95%乙醇:(2ml每1mlTRIPURE 用量)
振蕩,置室溫20min
離心: 7500g,5 min,4 度,棄上清
重復(fù)0.3M鹽酸胍/95%乙醇步2 次
沉淀中加入100%乙醇 2ml
充分振蕩混勻,置室溫20 min
離心: 7500g,5min,4度,棄上清吹干沉淀

1%SDS溶解沉淀
離心:10000g,10min,4度
取上清-20 度保存(或可直接用于WESTERN BLOT)
存在的問題:加入1%SDS 后沉淀不溶解,還是很大的一塊,4 度離心后又多了白色沉定,SDS 結(jié)晶?測(cè)
濃度,含量才1mg/ml左右。
解決:提蛋白試劑盒,另外組織大小適中,要碎,立即加2X BUFFER,然后煮5-10分鐘,效果很好的。
四、
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、
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(fù)離心5min
lysis buffer:urea np-40 ampholine 2-me pvp-40

六、
蛋白質(zhì)樣品制備(sigma)
秧苗蛋白質(zhì)樣品的提取按Davermal 等(1986)的方法進(jìn)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
氯乙酸(丙酮配制,含10mM 即0.07%β-巰基乙醇),混勻,-20℃沉淀1 小時(shí),4℃,15000 r/min離心15
min,棄上清,沉淀復(fù)溶于1.5ml冷丙酮(含10 mMβ-巰基乙醇),再于-20℃沉淀1 小時(shí),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(diào)) 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(dòng)幾次,28度 (溫度低,高濃度的尿素會(huì)讓溶液結(jié)冰)16000 r/min離心15 min,離心力越大時(shí)間長(zhǎng)一點(diǎn)
越好!上清即可上樣電泳?;蛘?70 度保存

七、
植物根中蛋白質(zhì)的抽?。╬henol)
(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(zhì)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(xì)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(guān) 于 蛋 白 質(zhì)

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(xì)胞線粒體蛋白,而不適用于線粒體功能檢測(cè)