请教：蛋白浓缩方法
请教各位高手，我想将细胞培养上清中的蛋白浓缩10倍，但要确保最小的蛋白变性与蛋白丢失，应该采用什么方法比较好？请不吝赐教，谢谢！
可以用：超虑；盐析；过离子柱等等，看你的试验条件定咯
蛋白浓缩方法基本有：
丙酮沉淀法；免疫沉淀法；三氯醋酸沉淀法；硫酸铵沉淀法；（低温）有机溶剂沉淀法；聚乙二醇沉淀法；超滤法；透析法；离子交换层析和冷冻干燥法……
1，丙酮沉淀法；三氯醋酸沉淀法 试验要求的仪器简单，但是常常导致蛋白质变性。
2，免疫沉淀法：得有特异性抗体！
3，硫酸铵沉淀法：利用高浓度盐将蛋白质析出（盐析）
选择硫酸按是因为：盐析有效性，pH范围广，溶解度高，溶液散热少，经济！
4，（低温）有机溶剂沉淀法：强调低温（0-4度以下）是因为10度时蛋白会在有机溶剂中变性，可用乙醇，丙酮……注意：Mg2+离子，pH值
5，聚乙二醇沉淀法：使用PEG时旨在个别情况下才会是蛋白质稍有变性！他溶解是散热低，形成沉淀的平衡时间短，通常达到30%时蛋白质就会达到最大量的沉淀！
6，超滤法：主要针对小体积蛋白质溶液（几ml）此法更不易引起变性！不过得有浓缩器，不是哪个实验室都有的！
7，透析法：主要用于更换蛋白质的缓冲液！的有透析袋！不需要特殊的仪器！
8，离子交换层析：可用阴离子交换树脂进行浓缩！
9，冷冻干燥法：在冷冻状态下让扬品种的液体升华
具体你的实验应该是做一种分泌蛋白吧？
浓缩后要干什么，需要活性蛋白么？
实验室设备如何，导师经费如何？
这些都要考虑！！
我用的是硫酸铵沉淀法，物廉价美。
用4度饱和硫酸铵等体积加入收集的上清中， 以30000g的速度离心30分钟，弃上清，加入你所需体积的缓冲液体，在震荡器上使沉淀溶解，再在10000g下离心10分钟取上清分装保存于-80度即可
关于蛋白浓缩，我认为最好的方法是真空冷冻干燥法，此法简便，蛋白质几乎无变性。
lhydy wrote:
请教各位高手，我想将细胞培养上清中的蛋白浓缩10倍，但要确保最小的蛋白变性与蛋白丢失，应该采用什么方法比较好？请不吝赐教，谢谢！
不知你何种用途，最简单的浓缩我们常用蔗糖或PEG包埋。
一个简便的方法你可以试试：找一透析袋，底部扎紧，袋口扎一去底的塑料或玻璃试管，将待浓缩的液体从管口灌入透析袋中，将整个装置挂在冰箱中，或者用电风扇吹，液体干后可再继续加入，直至样品浓缩至所需体积。如必要，可事先将待浓缩液用蒸馏水或去离子水透析以去盐分，然后再按上述方法浓缩，这样可以避免浓缩后盐份过高。此法简便易行，我们实验室常用此法浓缩。浓缩后的液体可以用吸管从试管口吸出，而且透析袋只要不破裂，可以反复使用。
可以用millipore 公司的amicon ultra－15 cenfilter unitetrifugal units。每个30多元，但是效果非常好。只需要将培养上清加入，然后高速离心后即可直接得到无菌的浓缩液。
对于新的透析袋有化学杂质，需要处理，为了去除这些杂质，通常我们用10mmol/L的碳酸氢钠，1mmol/L的EDTA溶液煮沸30min，之后用双蒸水充分冲洗，之后放在EDTA溶液4度保存！防止微生物污染！！
lrmoon介绍的方法听起来很好，能否具体介绍一下，谢谢！
Millipore's products are easy to use, but it intends to be blocked if you are using raw cell extracts or supernatant of culture media especially you want to collect small MW proteins. I once used a MW cuting 3000's, but I can only concentrate my samples 2-3 times before it was totally blocked. Basicly, it is a centrifuge tube with a membrane, a little bit like PCR purification kits, except protein is not binding with the membrane. The use of membrane is to separate the residue molecular from the filtrate molecular by molecular weight. You can choose different memberane by the MW cutting (from 3000 to 100000) hence concentrate your target compounds in the sample. It can also be used to desalt and so on.
thanks!!
冷冻干燥或peg20000都可以
amicon ultra－15 cenfilter unitetrifugal units可以反复用吗
From the instrument, they didn't say it can be reused. Acturally, I did call the supplier once and they also said it is not suggest to reuse because the membrane will be more or less blocked. And also, you can not wash the membrane very clean to avoid to bring contaminants from last sample. But I personally reused two tubes 4 time and the results are, be honest, quite good. You may remember to soak the tube in ddH2O after use otherwise, the membrane will dry and damaged.
amicon ultra－15 cenfilter unitetrifugal units 30元，
在哪里买的？
南京的代理商怎么告诉我要100块一个？
我也想做蛋白质的浓缩和脱盐,但蛋白质经亲和层析后,我想用来做双相电泳,但由于洗脱下来的蛋白质有盐,而且比较稀释(2-5ml),因此我想用milipore的柱子来浓缩和脱盐,但我现在还不知道他们的分子量范围到底多大,
请问我该选择哪一种柱子?国内哪个公司直接代理?我在重庆,重庆有代理的吗？
Well, there are two main categories. One is Amicon Ultra-15 Centrifugal Filter Units, the other is Amicon Ultra-4 Centrifugal Filter Units. 15 means the initial sample volume should be less 15 mL, the 4 means, of course, the initial sample volume should be no more than 4 mL. There maybe some differents between 15 and 4 in price because the larger tube volume, the larger membrane size. So, the 15 one should be a little bit expencive than 4, isn't it? You may want to ask the suppliers how many tubes per pack and also consider whether your centrifuge bucket can hold it (normally it should no problem, but better ask before you buy!). There are 2, 8, 24, and 96 tubes per pack. If you don't know the protein size, suggest you choose the most safe one: Amicon Ultra-4 5K Ultracel-PL memb 2/pk or 8/pk and it could be fixed into 10 mL centrifuge tube. Good luck.
Thank you!what mean is MW cutting from 3000 to 100000,for example, whether MW cutting 3000 means the proteins with MW more than 3000 are concentated and desalted?
That means the membrane cut-off is 3000. There is a formular to convert the cut-off to real molecular weigh. I forgot the formula, but 3000 cut-off is already very small. If I am not wrong, it converts to MW around several hundrads (not accurate here, sorry).
Yeah, you are right. If the MW higher than, (let's use 3000 to represent here), 3000, then it will not go through the membrane but left in the residue part. Small molecules, like sodium Na+, will go through the membrane to the filtrate part. So that, your sample is concentrated and at the same time desalted. If you choose membrane cut-off, let's say, 100000, than some proteins with small MW will also go through the membrane. That means, if you know your target protein's MW, you can choose a suitable membrane to separate the protein you want form others, isn't it? But of course, the shape of a molecule may also effect the filtration efficiency. If a molecule with large MW, theriatically, it will stay in the solution. But, if its shape is rod, than, maybe
some of them will also go through the membrane. So, I suggest that you could use the smallest one. I used 3000s one before, almost everything (I mean proteins) stays in the solution. Are we clear here? Good luck.
Thank you very much!!
cpuiris wrote:
amicon ultra－15 cenfilter unitetrifugal units 30元，
在哪里买的？
南京的代理商怎么告诉我要100块一个？
此种方法就是超滤浓缩，使用方式有的是离心，有的是加压，有的二者皆用。除了amicon之外，我知道赛多利斯也是很有名的供应厂家，就是做天平很出名的那家德国公司。超滤浓缩这种方式的确太方便了，而且除了有浓缩作用外，还有脱盐功能，速度又快，比传统的透析好多了，实验室用和生产用型号都能找到。不过据介绍，不能反复用！而且价格大家也知道了，30元左右，一般一个包装25个，100个，单个还不知道卖不卖。根据中国国情，呵呵，我建议没经费的实验室还是不要考虑啦；如果时间不紧张，不怕花几天时间，用25元/米的透析袋一样可以达到效果。
多谢各位高手的指教，能再请问一下用透析袋和PEG浓缩后，浓缩液中各种离子浓度是否会明显增高而影响进一步的实验？
谢谢！
我想用冷冻干燥法浓缩细胞上清，但听说并不是直接将高稀释的上清进行干燥，而先将上清浓缩一下比如用超滤离心，在进行干燥为好，是这样吗？另外，上清吸出后放在－70保存，日后在浓缩，蛋白应该不会降解把？
冷冻干燥的体积当然越小越好了，如果你的蛋白很稳定那-70度应该没有什么问题的，我觉得冷冻干燥时间长，如果量不大直接用沉淀的方法浓缩就可以了。浓缩的目的是使体积小这样快，而体积很大的话冷冻
干燥慢，如果你的冷冻干燥机的真空、泵很好，那么体积大也没有关系，但是要保证你的液体的高度别超过2cm那其实也是很快的，所以关键看你样品的体积和你机器的好坏了。
请教：蛋白浓缩方法
请教各位高手，我想将细胞培养上清中的蛋白浓缩10倍，但要确保最小的蛋白变性与蛋白丢失，应该采用什么方法比较好？请不吝赐教，谢谢！
可以用：超虑；盐析；过离子柱等等，看你的试验条件定咯
蛋白浓缩方法基本有：
丙酮沉淀法；免疫沉淀法；三氯醋酸沉淀法；硫酸铵沉淀法；（低温）有机溶剂沉淀法；聚乙二醇沉淀法；超滤法；透析法；离子交换层析和冷冻干燥法……
1，丙酮沉淀法；三氯醋酸沉淀法 试验要求的仪器简单，但是常常导致蛋白质变性。
2，免疫沉淀法：得有特异性抗体！
3，硫酸铵沉淀法：利用高浓度盐将蛋白质析出（盐析）
选择硫酸按是因为：盐析有效性，pH范围广，溶解度高，溶液散热少，经济！
4，（低温）有机溶剂沉淀法：强调低温（0-4度以下）是因为10度时蛋白会在有机溶剂中变性，可用乙醇，丙酮……注意：Mg2+离子，pH值
5，聚乙二醇沉淀法：使用PEG时旨在个别情况下才会是蛋白质稍有变性！他溶解是散热低，形成沉淀的平衡时间短，通常达到30%时蛋白质就会达到最大量的沉淀！
6，超滤法：主要针对小体积蛋白质溶液（几ml）此法更不易引起变性！不过得有浓缩器，不是哪个实验室都有的！
7，透析法：主要用于更换蛋白质的缓冲液！的有透析袋！不需要特殊的仪器！
8，离子交换层析：可用阴离子交换树脂进行浓缩！
9，冷冻干燥法：在冷冻状态下让扬品种的液体升华
具体你的实验应该是做一种分泌蛋白吧？
浓缩后要干什么，需要活性蛋白么？
实验室设备如何，导师经费如何？
这些都要考虑！！
我用的是硫酸铵沉淀法，物廉价美。
用4度饱和硫酸铵等体积加入收集的上清中， 以30000g的速度离心30分钟，弃上清，加入你所需体积的缓冲液体，在震荡器上使沉淀溶解，再在10000g下离心10分钟取上清分装保存于-80度即可
关于蛋白浓缩，我认为最好的方法是真空冷冻干燥法，此法简便，蛋白质几乎无变性。
lhydy wrote:
请教各位高手，我想将细胞培养上清中的蛋白浓缩10倍，但要确保最小的蛋白变性与蛋白丢失，应该采用什么方法比较好？请不吝赐教，谢谢！
不知你何种用途，最简单的浓缩我们常用蔗糖或PEG包埋。
一个简便的方法你可以试试：找一透析袋，底部扎紧，袋口扎一去底的塑料或玻璃试管，将待浓缩的液体从管口灌入透析袋中，将整个装置挂在冰箱中，或者用电风扇吹，液体干后可再继续加入，直至样品浓缩至所需体积。如必要，可事先将待浓缩液用蒸馏水或去离子水透析以去盐分，然后再按上述方法浓缩，这样可以避免浓缩后盐份过高。此法简便易行，我们实验室常用此法浓缩。浓缩后的液体可以用吸管从试管口吸出，而且透析袋只要不破裂，可以反复使用。
可以用millipore 公司的amicon ultra－15 cenfilter unitetrifugal units。每个30多元，但是效果非常好。只需要将培养上清加入，然后高速离心后即可直接得到无菌的浓缩液。
对于新的透析袋有化学杂质，需要处理，为了去除这些杂质，通常我们用10mmol/L的碳酸氢钠，1mmol/L的EDTA溶液煮沸30min，之后用双蒸水充分冲洗，之后放在EDTA溶液4度保存！防止微生物污染！！
lrmoon介绍的方法听起来很好，能否具体介绍一下，谢谢！
Millipore's products are easy to use, but it intends to be blocked if you are using raw cell extracts or supernatant of culture media especially you want to collect small MW proteins. I once used a MW cuting 3000's, but I can only concentrate my samples 2-3 times before it was totally blocked. Basicly, it is a centrifuge tube with a membrane, a little bit like PCR purification kits, except protein is not binding with the membrane. The use of membrane is to separate the residue molecular from the filtrate molecular by molecular weight. You can choose different memberane by the MW cutting (from 3000 to 100000) hence concentrate your target compounds in the sample. It can also be used to desalt and so on.
thanks!!
冷冻干燥或peg20000都可以
amicon ultra－15 cenfilter unitetrifugal units可以反复用吗
From the instrument, they didn't say it can be reused. Acturally, I did call the supplier once and they also said it is not suggest to reuse because the membrane will be more or less blocked. And also, you can not wash the membrane very clean to avoid to bring contaminants from last sample. But I personally reused two tubes 4 time and the results are, be honest, quite good. You may remember to soak the tube in ddH2O after use otherwise, the membrane will dry and damaged.
amicon ultra－15 cenfilter unitetrifugal units 30元，
在哪里买的？
南京的代理商怎么告诉我要100块一个？
我也想做蛋白质的浓缩和脱盐,但蛋白质经亲和层析后,我想用来做双相电泳,但由于洗脱下来的蛋白质有盐,而且比较稀释(2-5ml),因此我想用milipore的柱子来浓缩和脱盐,但我现在还不知道他们的分子量范围到底多大,
请问我该选择哪一种柱子?国内哪个公司直接代理?我在重庆,重庆有代理的吗？
Well, there are two main categories. One is Amicon Ultra-15 Centrifugal Filter Units, the other is Amicon Ultra-4 Centrifugal Filter Units. 15 means the initial sample volume should be less 15 mL, the 4 means, of course, the initial sample volume should be no more than 4 mL. There maybe some differents between 15 and 4 in price because the larger tube volume, the larger membrane size. So, the 15 one should be a little bit expencive than 4, isn't it? You may want to ask the suppliers how many tubes per pack and also consider whether your centrifuge bucket can hold it (normally it should no problem, but better ask before you buy!). There are 2, 8, 24, and 96 tubes per pack. If you don't know the protein size, suggest you choose the most safe one: Amicon Ultra-4 5K Ultracel-PL memb 2/pk or 8/pk and it could be fixed into 10 mL centrifuge tube. Good luck.
Thank you!what mean is MW cutting from 3000 to 100000,for example, whether MW cutting 3000 means the proteins with MW more than 3000 are concentated and desalted?
That means the membrane cut-off is 3000. There is a formular to convert the cut-off to real molecular weigh. I forgot the formula, but 3000 cut-off is already very small. If I am not wrong, it converts to MW around several hundrads (not accurate here, sorry).
Yeah, you are right. If the MW higher than, (let's use 3000 to represent here), 3000, then it will not go through the membrane but left in the residue part. Small molecules, like sodium Na+, will go through the membrane to the filtrate part. So that, your sample is concentrated and at the same time desalted. If you choose membrane cut-off, let's say, 100000, than some proteins with small MW will also go through the membrane. That means, if you know your target protein's MW, you can choose a suitable membrane to separate the protein you want form others, isn't it? But of course, the shape of a molecule may also effect the filtration efficiency. If a molecule with large MW, theriatically, it will stay in the solution. But, if its shape is rod, than, maybe
some of them will also go through the membrane. So, I suggest that you could use the smallest one. I used 3000s one before, almost everything (I mean proteins) stays in the solution. Are we clear here? Good luck.
Thank you very much!!
cpuiris wrote:
amicon ultra－15 cenfilter unitetrifugal units 30元，
在哪里买的？
南京的代理商怎么告诉我要100块一个？
此种方法就是超滤浓缩，使用方式有的是离心，有的是加压，有的二者皆用。除了amicon之外，我知道赛多利斯也是很有名的供应厂家，就是做天平很出名的那家德国公司。超滤浓缩这种方式的确太方便了，而且除了有浓缩作用外，还有脱盐功能，速度又快，比传统的透析好多了，实验室用和生产用型号都能找到。不过据介绍，不能反复用！而且价格大家也知道了，30元左右，一般一个包装25个，100个，单个还不知道卖不卖。根据中国国情，呵呵，我建议没经费的实验室还是不要考虑啦；如果时间不紧张，不怕花几天时间，用25元/米的透析袋一样可以达到效果。
多谢各位高手的指教，能再请问一下用透析袋和PEG浓缩后，浓缩液中各种离子浓度是否会明显增高而影响进一步的实验？
谢谢！
我想用冷冻干燥法浓缩细胞上清，但听说并不是直接将高稀释的上清进行干燥，而先将上清浓缩一下比如用超滤离心，在进行干燥为好，是这样吗？另外，上清吸出后放在－70保存，日后在浓缩，蛋白应该不会降解把？
冷冻干燥的体积当然越小越好了，如果你的蛋白很稳定那-70度应该没有什么问题的，我觉得冷冻干燥时间长，如果量不大直接用沉淀的方法浓缩就可以了。浓缩的目的是使体积小这样快，而体积很大的话冷冻
干燥慢，如果你的冷冻干燥机的真空、泵很好，那么体积大也没有关系，但是要保证你的液体的高度别超过2cm那其实也是很快的，所以关键看你样品的体积和你机器的好坏了。