KOSEN 한인과학기술자네트워크

만일 약한 농도의 HCL로 추출 했다면 그냥 TCA down후 diethyl ether로 2번 washing한후 말려서 걸면 될듯합니다. 아니면 약한농도의 NaOH로 서서히 중화(pH paper로 확인)시킨후 역시 TCA down후 diethyl ether로 2번 washing한후 말려서 걸면 될듯합니다. >제가 하는 실험에서 제가 하는 실험에서 HCl로 추출한 단백질을 > >전기영동을 해야하는데 염산때문에 문제가 발생해서 precipitaion이나 > >dialysis를 하려고 하는데 혹시 염산으로 추출한 단백질을 > >다시 TCA로 Precipitation하면 > >전기영동을 하여 웨스턴을 하는데 문제가 없을까요? > >오늘 실험을 하려다가 갑자기 이런 의문이 생겨서 글을 올립니다.

차이는 모르겠습니다. 제가 대학원 때는 랩에서Diethyl ether를 acetone보다 선호했습니다. 냄새는 좀 나는데 TCA제거에 더 효과적이라서 그걸썼는지는 잘 모르겠군요. >제가 하는 실험에서 제가 하는 실험에서 HCl로 추출한 단백질을 > >전기영동을 해야하는데 염산때문에 문제가 발생해서 precipitaion이나 > >dialysis를 하려고 하는데 혹시 염산으로 추출한 단백질을 > >다시 TCA로 Precipitation하면 > >전기영동을 하여 웨스턴을 하는데 문제가 없을까요? > >오늘 실험을 하려다가 갑자기 이런 의문이 생겨서 글을 올립니다.

Washing시 ethanol:ether (1:1 v/v)를 사용함을 주의하세요. 단순히 ether만을 사용하면 TCA제거가 잘 안됩니다. 아래는 web에서 제가 쓴 protocol에 가장 가까운것을 따온 겁니다. link: http://www.science.mcmaster.ca/biochem/faculty/andrews/lab/projects/methodsandprograms/labman/tca_precipitation_of_proteins.html TCA precipitation of proteins. The most common reason that we TCA precipitate proteins is to concentrate them prior to SDS-PAGE. After SDS-PAGE the proteins are visualized either by staining the gel or by western blotting. This protocol is for this application of TCA precipitation. There are slight differences in the protocol if when used for other purposes. These are noted at the end. Trichloroacetic acid (TCA) is very nasty! Remember the point here is to denature and precipitate proteins and YOU are made of proteins. It is very important to wear proper protective outerwear when using TCA. Minimally you need a lab coat and safety glasses and latex gloves. Remember - A small puncture in your gloves can lead to a nasty burn. You should prepare some 100 mM sodium bicarbonate buffer in case you have a spill or get some TCA on an unprotected area of skin by accident. If you get any TCA on you or your clothes douse with 100 mM sodium bicarbonate buffer and then wash with lots of water. TCA precipitate the chilled products by adding 2 volumes of ice cold 20% TCA, vortex, and incubate from 10 mins to several hours on ice. Alternatively you can use 1/2 volume of 50% TCA (w/v) but obviously 50% TCA is more hazardous than 20%. The idea is to end up with ~15% TCA as the final concentration. Spin out precipitate 15 to 20 seconds in microfuge, aspirate sup., add 400 ul ice cold ethanol:ether (1:1 v/v) spin again and aspirate sup. Ether is also hazardous - carefully check any bottle of Ether for crystals. If there is any crystalline precipitate in a bottle of ether do not use it. Carefully return it to the flammables cupboard and arrange for disposal. Let the pellets dry in a fumehood for 5-10 minutes. Add 1X SDS-PAGE loading Buffer. If you use loading buffer containing bromphenol blue and it turns yellow then there is still some acid left in your precipitate. You can neutralize this with 1M Tris of whatever pH you use for the stacking gel. If you use loading buffer with red dye then dissolve your pellets in 0.1 M Tris pH 8.9 (add a volume equal to 1/2 the amount you want to load in a lane on the gel). Vortex, and if the precipitate does not go into solution immediately incubate at 37øC for half an hour vortexing every 5-10 minutes. NOTES: If you want to immunoprecipitate the protein (for example when using in vitro translation products and an antibody that recognizes the denatured protein or if you want to immunoprecipitate your protein without any putative binding partners) dissolve your pellets in 100 ul of 0.1 M Tris pH 8.9, 1% SDS. Vortex, and if the precipitate does not go into solution immediately incubate at 37øC for half an hour vortexing every 5-10 minutes. If that doesn't work heat the dissolved samples in boiling bath for 2-4 minutes. Dilute samples with 20 volumes of 1 X TX-SWB to take up all excess SDS into Triton micelles (final Triton conc is approx. 1%, final SDS conc is approximately .05%.) Add antiserum and proceed as for previous immunoprecipitation protocol starting with step 3. If you are TCA precipitating reticulocyte lysate make sure you don't try to precipitate more than 1 ul or you will get concrete. Ammonium sulphate precipitation is probably better for retic. >제가 하는 실험에서 제가 하는 실험에서 HCl로 추출한 단백질을 > >전기영동을 해야하는데 염산때문에 문제가 발생해서 precipitaion이나 > >dialysis를 하려고 하는데 혹시 염산으로 추출한 단백질을 > >다시 TCA로 Precipitation하면 > >전기영동을 하여 웨스턴을 하는데 문제가 없을까요? > >오늘 실험을 하려다가 갑자기 이런 의문이 생겨서 글을 올립니다.