JoVE Science Education
Biochemistry
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JoVE Science Education Biochemistry
Dialysis: Diffusion Based Separation
  • 00:00Overview
  • 00:40Principles of Dialysis
  • 02:41Dialysis Procedure
  • 03:51Applications
  • 05:07Summary

透析: 基于扩散的分离

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Overview

透析是生物化学中用于分离分子扩散的常用技术。在这个过程中, 透膜允许某些分子基于大小的运动。这种方法可以用于去除缓冲, 称为脱盐, 或交换缓冲分子或离子从蛋白质溶液.

本视频介绍了透析的原理和一般程序. 和 #160; 综述了几种透析方法的应用, 包括去除离心后的梯度试剂, 去除膜蛋白后的洗涤剂通过改变溶液环境来提取和重组蛋白质.

生物化学样本通常具有较高的缓冲浓度, 可以破坏下游的处理和分析。透析是一种常见的, 廉价的技术用于分离分子的基础上扩散。该方法利用半膜, 允许基于大小的某些成分的移动。这个视频将显示透析的概念, 一个一般的程序, 以及它在生物化学中的一些用途.

透析的最重要的方面是半膜, 它的毛孔会施加分子量的限制, 使分子低于一定的大小才能通过。例如, 10k 膜通常会保留大于 10 kilodaltons 的分子。然而, 分子量的截止不是一个离散或精确的边界。该膜通常含有广泛的孔隙大小, 所以在截止距离附近的一小部分分子可能会丢失.

由于分子量的分界是使用球状蛋白质定义的, 类似质量的线性分子, 如 DNA 或 RNA, 可能会滑过。膜通常选择一个半到三分之一的分子量的期望分子.

执行过程中, 将一个样本放入膜中, 这反过来又增加了大量的溶液, 称为透析液。随着时间的推移, 较小的分子将在样品和透析液之间自由扩散, 而更大的生物大分子则被保存在体内。透析是一个缓慢的过程。这是常见的, 让它运行过夜, 甚至跨越多天.

如果透析液是纯净水, 总的缓冲浓度会降低, 这一过程称为脱盐。如果解决方案包含其他小粒子, 一些将进入样本, 导致缓冲交换。因为透析是一个平衡过程, 所以透析液可以被多次刷新以进一步取代小分子。完成该过程后, 示例将 re-collected 以进行进一步处理.

现在, 您和 #39; 我已经看到了透析的基本知识, 让我们 #39; 我们来看看一般的程序.

在开始操作前, 膜在透析液中碳纤维。这使得它更容易使用, 并删除任何防腐剂。一旦准备好, 样品收集, 通常与注射器, 然后添加到透析容器。这可以是裸油管, 或包含在一个卡带。过量的空气从透析装置中除去, 以使样品和 #39 的表面积最大化。然后将设置放入透析液中, 搅拌以最大限度地扩散。它应该漂浮不抑制搅动.

透析液在相应的时间间隔内改变, 以达到样品与透析液的平衡。在最后的变化后, 反应通常会在一夜之间运行。在足够的时间段后, 将从卡带中取出无缓冲或交换的样品。一旦收集, 根据实验的性质, 可以对样品进行分析或进一步处理.

现在我们和 #39; 我看了一个一般的透析程序, 让和 #39; 我们看到了这种技术用于生物化学的一些方法.

密度梯度是分离复杂生物样品的常用方法。这个概念依赖于小颗粒的分布, 通常是蔗糖或氯化铯离子。一旦完成, 这些试剂通常需要删除之前, 收集的样本可以处理。透析可以利用纯化的样品进行未来的分析.

在细胞和 #39 的脂质双层中发现某些蛋白质, 通常通过散脂质囊的方法研究它们。首先用洗涤剂提取蛋白质和脂质。透析可以用来慢慢去除洗涤剂, 形成 proteoliposomes.

纯化后, 某些蛋白质错误或变性, 导致功能丧失。导致结构变化的化合物可以通过透析去除, 导致功能性分析的改革.

您和 #39; 我刚刚看过朱庇特和 #39 的透析视频。您现在应该了解这种扩散方法, 一个简单的实验过程, 以及使用此技术.

感谢收看!

Procedure

Dialysis is a common technique used in biochemistry for separating molecules based on diffusion. In this procedure, a semipermeable membrane allows the movement of certain molecules based on size. This method can be applied to the removal of buffer, known as desalting, or exchanging buffer molecules or ions from a protein solution. This video covers the principles of dialysis along with a general procedure.  Several applications of dialysis are reviewed, including the removal of gradient reagents following ultracentrifugation, removing detergent after a membrane protein extraction, and the reconstitution of proteins by changing the solution environment.

Disclosures

No conflicts of interest declared.

Transcript

Biochemical samples typically have high buffer concentrations that can disrupt downstream processing and analysis. Dialysis is a common, inexpensive technique used to separate molecules based on diffusion. The method utilizes a semi-permeable membrane that allows the movement of certain components, based on size. This video will show the concepts of dialysis, a general procedure, and some of its uses in biochemistry.

The most important aspect of dialysis is a semi-permeable membrane, which has pores that impose a molecular weight cut-off, allowing molecules below a certain size to pass through. For example, a 10k membrane will generally retain molecules larger than 10 kilodaltons. However, the molecular weight cutoff is not a discrete or precise boundary. The membrane typically contains a broad range of pore sizes, so a small fraction of molecules near the cutoff may be lost.

Since the molecular weight cutoff is defined using globular proteins, linear molecules of similar mass, like DNA or RNA, may slip through. Membranes are typically chosen one half to one third the molecular weight of the desired molecule.

To perform the procedure, a sample is placed into the membrane, which is in turn added to a large volume of solution, called the dialysate. Over time, smaller molecules will diffuse freely across the membrane between the sample and the dialysate, while the larger biomolecules are held within. Dialysis is a slow process. It is common to allow it to run overnight, or even across multiple days.

If the dialysate is pure water, the overall buffer concentration will decrease, a process known as desalting. If the solution contains other small particles, some will move into the sample, leading to buffer exchange. Because dialysis is an equilibrium process, the dialysate can be refreshed multiple times to further displace small molecules. Once the process is complete the sample is re-collected for further processing.

Now that you’ve seen the basics of dialysis, let’s take a look at a general procedure.

Before beginning the procedure, the membrane is presoaked in dialysate. This makes it easier to use, and removes any preservatives. Once ready, the sample is collected, typically with a syringe and is then added to the dialysis container. This can be bare tubing, or contained within a cassette. Excess air is removed from the dialysis setup to maximize the sample’s surface area with the membrane. The setup is then placed into the dialysate with stirring to maximize the diffusion. It should float to not inhibit stirring.

The dialysate is changed at relevant intervals as equilibrium between sample and dialysate is reached. After the last change, the reaction is typically left to run overnight. After a sufficient time period, the buffer-free or -exchanged sample is removed from the cassette. Once collected, the sample can be analyzed or further processed, depending on the nature of the experiment.

Now that we’ve looked at a general dialysis procedure, let’s see some of the ways this technique is used in biochemistry.

Density gradients are a common way to separate complex biological samples. This concept relies on the distribution on small particles, typically sucrose or cesium chloride ions. Once complete, these reagents typically need to be removed before the collected sample can be processed. Dialysis makes it possible to utilize the purified sample for future analysis.

Certain proteins are found within a cell’s lipid bilayer, and are usually studied by interspersing them into spherical lipid vesicles known as liposomes. The proteins and lipids are first extracted with a detergent. Dialysis can be used to slowly remove the detergent, forming proteoliposomes.

After purification, some proteins are misfolded, or denatured, leading to a loss in functionality. The compounds that cause these changes in structure can be removed with dialysis, leading to the reformation of functioning analytes.

You’ve just watched JoVE’s video on dialysis. You should now understand this diffusion-based method, a simple experimental procedure, and the use of this technique.

Thanks for watching!

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JoVE Science Education Database. JoVE Science Education. Dialysis: Diffusion Based Separation. JoVE, Cambridge, MA, (2023).

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