Protein Analysis: Customized Isolation and Purification
Natural and Recombinant Proteins
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| Isolation and Purification |
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Natural source proteins are proteins derived from biological substances, such as urine, saliva, blood, and other tissues. Because natural source
protein purification is more complicated than most life science services, purification procedures must be developed specifically for a given protein. Such a process requires significant interaction between GBS and its customers. GBS typically purifies these types of proteins via single or multiple chromatography separations. For standard chromatographic fractionations, a Waters HPLC system is used, but often batch or gravity-based chromatography, or non-chromatographic purification methods (e.g. organic or salt precipitation, ultrafiltration) are also required.
The typical process for developing a formal purification quote progresses as follows:
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The client provides GBS with as much information about the target protein as possible. |
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GBS scientists review the information, clarify the specifics via email communication with the client, and review the pertinent scientific literature |
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Using input from these sources, GBS scientists develop the purification procedure with the highest probability of success for the target protein and a formal quote is prepared. |
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However, if methodological uncertainties existed after the thorough background investigation, GBS may suggest and quote some preliminary key analyses to eliminate these uncertainties prior to performing the purification work. The recommended preliminary work is offered to provide the client with the highest possible confidence that their starting material will be processed with the greatest chance of success. |
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After the results of the initial analyses have been returned to the client, a quote for preparative isolation of the target protein, based on information gained from the preceding work, is developed by GBS. The preliminary work is not mandatory, but generally provides the most cost efficient means of purifying challenging proteins. |
Since a natural source target protein is not tagged, GBS requires that all customers provide a means or method of identifying their protein(s) of interest. The method of identification may be a biochemical attribute (isoelectric point, molecular weight), a biochemical (enzymatic) assay, or an appropriate antibody (for western blots or ELISA). If a biochemical assay is chosen for the detection method, the customer must also provide a complete protocol with a list of necessary reagents.
PLEASE NOTE: The method of target protein identification generally comprises a large portion of the total cost of the purification, so the choice should be carefully considered. Specifically, the number of steps and amount of labor involved in the detection method should be minimized if budgeting is tight, and attention to reagent costs is also important. Fees for biochemical assays and western blot identification are determined on a case-by-case basis.
Recombinant Proteins
GBS also purifies tagged recombinant proteins, a process that is less involved than the equivalent efforts with natural source proteins.
Specifically, GBS is equipped to isolate proteins with polyhistidine tags from
Escherichia coli lysates. During this procedure, a small aliquot of the lysate is removed for SDS PAGE analysis, a second lysate aliquot is centrifuged to separate soluble and insoluble fractions, and SDS PAGE analysis is used to determine how recombinant protein partitions between the two fractions. Then, the Ni affinity chromatography is applied to the appropriate fraction to isolate the recombinant product, using SDS PAGE to monitor the efficiency of the process. If the recombinant product still contains unacceptable levels of protein contaminants, additional chromatography steps or chromatography optimization are applied to improve purity. For additional cost, purified protein can be concentrated and desalted, and total protein and percent purity determined.
Recombinant proteins with other tags (GST fusion, etc) can be purified; however, because other recombinant chromatography systems are not routinely applied in-house at GBS, the costs will be greater.
Note: Because recombinant proteins vary considerably in their susceptibility to purification schemes, GBS cannot guarantee purification success. However, GBS scientists will work with customers to achieve the highest possible efficiency within existing constraints. Before contacting GBS, please visit the sample preparation page for tips on maximizing purification results.
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Buffer composition*
Sample complexity
Total protein content
Method of identification for protein
Type of protein (Recombinant or Natural Source)
For an explanation of any of these items, please see the
Sample Preparation Page.)
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For Natural Source Proteins
SDS PAGE gel image of peak
Purified protein (freeze dried unless otherwise indicated)
Chromatogram from LC run
For Recombinant Proteins
SDS PAGE gel images following the purification steps
Purified protein (freeze dried unless otherwise indicated)
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Custom Pricing** Contact GBS by
email,
contact form, or by downloading our
custom quote form.
*The customer is required to inform GBS if source material contains biological toxins, highly toxic reagents, or potentially infectious material, and if there are any special handling requirements.
Analysis of radioactive material is not supported.
*Customer is required to inform GBS if source material contains biological toxins, highly toxic reagents, or potentially infectious material, and if there are any special handling requirements.
Analysis of radioactive material is not supported.
**Final price quote includes procurement of necessary reagents for biochemical assays.
(Reagent delivery time may affect analysis report time.)