TCA 蛋白沉淀方法

1、100（w/v）三氯乙酸的配制方法： 500g三氯乙酸用227ml水来溶解，所得溶液即100三氯乙酸溶液。避光，4度保存。(preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, use gloves!).培养基上清直接电泳跑出来的条带经常很难看，可以TCA沉淀浓缩后跑电泳，一般表达量大于1mg/ml可以看到明显条带，这是我用的TCA沉淀方法，效果很好：1.菌液10000g,离心5分钟，收集表达上清。2.取500-1000ul上清于EP管中，加入1/9体积的100TCA，颠倒10次

2、混匀。3.样品置于冰浴中大于0.5小时，过夜效果更好。4.15000g,离心1020分钟,可见有棕黑色沉淀，倒掉上清，将EP管倒扣在吸水纸上轻轻控几下，除去残余在管口的液体。5.将EP管倒置于吸水纸长，37度烘箱1020分钟，待管底无明显液体残留，如果管壁还残留有液体，可以吸水纸吸掉。可以改成室温或用电吹风，关键是除去管底和管壁残余液体。6.15000g,离心1020分钟,用20ul枪头尽量吸去管底残余的液体，此步骤要快，不然沉淀容易散开，降低蛋白回收率，一般最多几ul或者没有，注意不要吸到沉淀。7.EP管倒置于吸水纸长，37度烘箱5分钟，确认管壁和管底没有液体残留。8.加入2050ul Lo

3、ading buffer，95度加热10nim，一般沉淀会自动溶解，如果不溶，用手指轻弹管壁或用20ul枪头轻轻吸打，注意整个操作尽量不要碰到管壁，因为管壁可能沾有残余TCA。如果蓝色的Loading buffer不变成黄色，说明残余TCA吸弃了干净，如果变黄，一般不影响电泳。此方法连丙酮洗这一步都省了，而且不影响电泳效果。或者第5步和第6步改为丙酮洗:5.加入200ul冰冷的丙酮，用手指轻弹EP管，洗去管底和管壁残余的TCA。6.15000g,离心1020分钟,倒掉上清，将EP管倒扣在吸水纸上轻轻控几下，除去残余在管口的液体。TCA-DOC For precipitation of very

4、 low protein concentration 1) To one volume of protein solution, add 1/100 vol. of 2% DOC (Na deoxycholate, detergent). 2) Vortex and let sit for 30min at 4oC. 3) Add 1/10 of Trichloroacetic acid (TCA) 100% vortex and let sit ON at 4oC (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bot

5、tleat 4oC.Be careful, use gloves!). 4) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see). OPTION: Wash pellet twice with one volume of cold acetone (acetone keep at 20oC

6、). Vortex and repellet samples 5min at full speed between washes. 5) Dry samples under vaccum (speed vac) or dry air. For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ;

7、titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.) Normal TCA To eliminate TCA soluble interferences and protein concentration 1) To a sample of protein solution add Trichloroacetic acid (TCA) 100% to get 13% final concentration. Mix and keep 5min 20oC and then

8、 15min 4oC; or longer time at 4oC without the 20oC step for lower protein concentration. Suggestion: leave ON if the protein concentration is very low. (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, use gloves!). 2) Spin 15min 4oC in microfuge at maximum speed

9、(15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see). 3) For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. (The presence of some TCA can give a yellow colour as a consequence of the acidification o

10、f the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.) Acetone Precipitation To eliminate acetone soluble interferences and protein concentration 1) Add to 1 volume of protein solution 4 volumes of cold acetone. Mix and keep at least 20min 20o

11、C. (Suggestion: leave ON if the protein concentration is very low). 2) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see). 3) Dry samples under vaccum (speed-vac) or dry

12、air to eliminate any acetone residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. Ethanol Precipitation Useful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS 1) Add to 1 volume of protein solution 9 volumes of cold Ethano

13、l 100%. Mix and keep at least 10min.at 20oC. (Suggestion: leave ON). 2) Spin 15min 4oC in microcentrifuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see). 3) Wash pellet with 90% cold ethanol (ke

14、ep at 20oC). Vortex and repellet samples 5min at full speed. 4) Dry samples under vaccum (speed vac) or dry air to eliminate any ethanol residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. TCA-DOC/Acetone Useful method to concentrate proteins and remove aceto

15、ne and TCA soluble interferences 1. To one volume of protein solution add 2% Na deoxycholate (DOC) to 0.02% final (for 100 l sample, add 1 l 2% DOC). 2. Mix and keep at room temperature for at least 15 min. 3. 100% trichloroacetic acid (TCA) to get 10% final concentration (preparation of 100% TCA: 4

16、54ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, use gloves!). 4. Mix and keep at room temperature for at least 1 hour. 5. Spin at 4oC for 10 min, remove supernatant and retain the pellet. Dry tube by inversion on tissue paper. 6. Add 200 l of ice cold acetone to TCA pellet. 7. Mix and kee

17、p on ice for at least 15 min. 8. Spin at 4oC for 10 min in microcentrifuge at maximum speed. 9. Remove supernatant as before (5), dry air pellet to eliminate any acetone residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. 10. (The presence of some TCA can giv

18、e a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.) Acidified Acetone/Methanol Useful method to remove acetone and methanol soluble interferences like SDS before IEF 1) Prepare acidif

19、ied acetone: 120ml acetone + 10l HCl (1mM final concentration). 2) Prepare precipitation reagent: Mix equal volumes of acidified acetone and methanol and keep at -20oC. 3) To one volume of protein solution add 4 volumes of cold precipitation reagent. Mix and keep ON at -20oC. 4) Spin 15min 4oC in mi

20、crofuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see). 5) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone or methanol residue (smell tubes). TCA-Ethanol Precipitation U

21、seful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS 1) Dilute 10-25l samples to 100l with H2O Add 100l of 20% trichloroacetic acid (TCA) and mix (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, use gloves!). 2) Leave in ice

22、 for 20min. Spin at 4oC for 15 min in microcentrifuge at maximum speed. 3) Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue (pellet may be difficult to see). 4) Wash pellet with 100l ice-cold ethanol, dry and resuspend in sample buffer. 5) In case there are trac

23、es of GuHCl present, samples should be loaded immediately after boiling for 7 min at 95C 6) (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.) PAGE prepTM Protein Clean-up and E