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蛋白质提取的方法总汇
双击自动滚屏 发布者:biohao 发布时间:2011-11-8 阅读:10241

、植物组织蛋白质提取方法(summer

1、根据样品重量(1g样品加入3.5ml提取液,可根据材料不同适当加入),准备提取液放在冰上。

2、把样品放在研钵中用液氮研磨,研磨后加入提取液中在冰上静置(3-4小时)。

3、用离心机离心8000rpm40min4℃11100rpm20min4℃

4、提取上清夜,样品制备完成。

蛋白质提取液:300ml

11Mtris-HClPH8 45ml

2、甘油(Glycerol75ml

3、聚乙烯吡咯烷酮(Polyvinylpolypyrrordone6g

这种方法针对SDS-PAGE,垂直板电泳!

 

、三氯醋酸—丙酮沉淀法

1、在液氮中研磨叶片

2、加入样品体积3倍的提取液在-20℃的条件下过夜,然后离心(4℃8000rpm以上1小时)弃上清。

3、加入等体积的冰浴丙酮(含0.07%β-巯基乙醇),摇匀后离心(4℃8000rpm以上1小时),然后真空干燥沉淀,备用。

4、上样前加入裂解液,室温放置30分钟,使蛋白充分溶于裂解液中,然后离心(15℃8000rpm以上1小时或更长时间以没有沉淀为标准),可临时保存在4℃待用。

5、用Brandford法定量蛋白,然后可分装放入-80℃备用。

药品:

提取液:含10%TCA0.07%β-巯基乙醇的丙酮

裂解液:2.7g尿素0.2gCHAPS溶于3ml灭菌的去离子水中(终体积为5ml),使用前再加入1MDTT65ul/ml

这种方法针对双向电泳,杂质少,离子浓度小的特点!当然单向电泳也同样适用,只是电泳的条带会减少!

 

、组织:肠黏膜(newinbio

目的:WESTERN BLOT检测凋亡相关蛋白的表达

应用TRIPURE提取蛋白质步骤:

1.含蛋白质上清液中加入异丙醇:(1.5ml1mlTRIPURE用量)

2.倒转混匀,置室温10min

3.离心:12000 g10min4度,弃上清

4.加入0.3M盐酸胍/95%乙醇:(2ml1mlTRIPURE用量)

5.振荡,置室温20min

6.离心: 7500g5 min4度,弃上清

7.重复0.3M盐酸胍/95%乙醇步2

8.沉淀中加入100%乙醇 2ml

9.充分振荡混匀,置室温20 min

10.离心: 7500g5min4度,弃上清吹干沉淀

11.1SDS溶解沉淀

12.离心:10000g10min4

13.取上清-20度保存(或可直接用于WESTERN BLOT

存在的问题:加入1SDS后沉淀不溶解,还是很大的一块,4度离心后又多了白色沉定,SDS结晶?测浓度,含量才1mg/ml左右。

解决:提蛋白试剂盒,另外组织大小适中,要碎,立即加2X BUFFER,然后煮510分钟,效果很好的。

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.

、植物材料:水稻苗,叶鞘,根(ynibcas

1200毫克样品置于冰上磨碎

2、加lysis buffer,离心,10000rpm4度,5min取上清

3、重复离心5min

lysis bufferurea   np-40  ampholine 2-me  pvp-40

、蛋白质样品制备(sigma

秧苗蛋白质样品的提取按Davermal等(1986)的方法进行。

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10mM0.07%β-巯基乙醇),混匀,-20℃沉淀1小时,4℃15000 r/min离心15 min,弃上清,沉淀复溶于1.5ml冷丙酮(10 mMβ-巯基乙醇),再于-20℃沉淀1小时,同上离心弃上清,(有必要再用80%丙酮(10 mMβ-巯基乙醇所得沉淀低温冷冻真空抽干。

按每mg干粉加入20μl(可调) UKS[9.5 M尿素,5mM碳酸钾,1.25%SDS0.5%DTT(二硫苏糖醇)2% Ampholine (Amersham Pharmacia Biotech IncpH3.5-10)6% Triton X-100]37℃温育30min,期间搅动几次,28度 (温度低,高浓度的尿素会让溶液结冰)16000 r/min离心15 min,离心力越大时间长一点越好!上清即可上样电泳。或者-70度保存

、植物根中蛋白质的抽取(phenol

(1) sample, 液氮研磨

(2) 1.5 ml centrifuge tube

(3) 1M KH2PO4+K2HPO4 700 ul

(4) 12000 rpm, 4, 10-15minite

(5) 取上层液,蛋白质就在里面

 

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. Immediately 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

、材料:细菌蛋白(puc18

用甲醇提取的,冻干后用缓冲液溶解的。样品缓冲液是一般的。其中含又2%SDS20mmol2-巯基乙醇。

、线粒体蛋白的提取 (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

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, 20029912825–12830

本方法只适用于提大鼠细胞线粒体蛋白,而不适用于线粒体功能检测

 
 

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