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This section will give you step-by-step procedures for purifying ß-galactosidase. In addition, it is designed to give you some background information for each of the different techniques in this purification strategy. For more detailed background information, and for information about other separation techniques, consult the resource materials in Appendix C. Separation Methods: As described in Chapter 2, there are various techniques used to separate proteins from one another. Each of these techniques is based on a particular property by which proteins differ from one another. Some properties of proteins that are used as a basis for separation techniques include:
The acronym CHASM is often used to summarize this list of properties. In order to purify a particular protein, it is usually necessary to use several separation techniques in a series.
ResolutionSeparation methods are often categorized according to the degree of resolution. Resolution refers to the resolving power of a method - its ability to separate (resolve) two proteins that are similar in properties. For example, a high resolution method that separates by size, is capable of separating two proteins that have very similar molecular weights, such as 50,000 daltons and 52,000 daltons. A low resolution method could only separate two proteins if they were very different in size such as 100,000 and 50,000 daltons. Ammonium sulfate precipitation is an example of a low resolution method while ion exchange chromatography is a high resolution method.
CapacitySeparation methods are often described as being either low or high capacity techniques. As the name implies, this characterization refers to how much volume the technique can handle. Although, in principle, any method can be scaled up to increase the capacity, a low capacity technique would be more difficult (or prohibitively expensive) to scale up. Some forms of column chromatography are more amenable to scale-up than others. For example, ion exchange can be scaled up to a high capacity method quite easily whereas gel filtration chromatography is more suited for smaller sample volumes.
Preparative or AnalyticalThe same separation method can be used for either a preparative or an analytical purpose. An "analytical" procedure is performed primarily to obtain information: for example, to analyze the results of a purification step. For analytical procedures, aliquots of the protein mixture are used and are generally thrown away after the analysis is complete. In a preparative step, the entire protein mixture, not an aliquot, is used for the purification step since the purpose is to separate the target protein from contaminants in a non-destructive manner.
Common Themes for Purification StrategiesAlthough each purification strategy is unique, there are some common themes. Generally, high capacity techniques with low resolution are used earlier in the purification strategy and higher resolution steps are used later in the process. Steps that result in dilution of the target protein sample are usually followed by steps that concentrate the volume of the sample. At some point, desalting and/or buffer exchange is usually necessary and either dialysis, gel filtration or ultrafiltration may be used for that purpose.
Overview of our Purification StrategyThe ß-galactosidase purification strategy used in this manual is outlined in Figure 4.1 in a flowchart format. As a first step, a crude extract will be made using sonication, followed by ammonium sulfate precipitation as a high capacity, low resolution step to remove some contaminating proteins and concentrate the volume of extract. Excess salt is then removed by dialysis, and the sample is loaded on an ion exchange column for a high resolution separation step. We will analyze the success of the purification by specific activity calculations, by SDS PAGE analysis and finally by Western Blotting.
Figure 4.1: Flow chart. Although we will stop the purification after the ion exchange step, this would probably not be the final step in most research or industrial situations. Even if the product was judged to be “pure”, the salt in the NTM buffer used for elution would probably be removed using ultrafiltration, tangential flow filtration or another “polishing” method appropriate to the scale of the purification process. If the product was to be used as a drug, more rigorous tests to determine purity would be used including HPLC methods, endotoxin testing, etc.
Aliquots and AssaysAliquots will be saved after each separation step before proceeding with the next step. Some aliquots will be used right away for enzyme assay to verify that the majority of the enzyme activity has been captured by the technique. The specific activity is usually determined after each purification step; however, it is possible to postpone protein assays until a more convenient time, as long as you save aliquots from each step. For example, you could begin dialysis before you complete the protein assay needed for specific activity determination of the AS pellet. However, do not start the dialysis until you know that the sample contains the majority of the enzyme activity. Some aliquots will be stored and saved for later use after all preparative purification procedures are completed. These aliquots will be used for SDS PAGE and HPLC (optional) as described in section 4. As you proceed through the purification procedures, the total mass of all proteins (as measured by the BioRad assay) should decrease since you are removing contaminating proteins at each step. The total enzyme activity will also decrease somewhat but should not decrease as dramatically since you are trying to keep as much of the target protein as possible. Therefore, the specific activity should increase as your protein becomes more pure.
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