Affinity chromatography nucleic acids

Affinity Chromatography Template Chromatography of Nucleic Acids and Proteins, Herbert Schott... 

In this chapter, we will survey the kinds of solid supports (substrates) and surface chemistries currently used in the creation of nucleic acid and protein microarrays. Which are the best supports and methods of attachment for nucleic acids or proteins Does it make sense to use the same attachment chemistry or substrate format for these biomolecules In order to begin to understand these kinds of questions, it is important to briefly review how such biomolecules were attached in the past to other solid supports such as affinity chromatography media, membranes, and enzyme-linked immxm-osorbent assay (ELISA) microtiter plates. However, the microarray substrate does not share certain unique properties and metrics with its predecessors. Principal among these are printing, spot morphology, and image analysis they are the subjects of subsequent chapters. 

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

Other methods that are related to affinity chromatography include hydrophobic interaction chromatography and thiophilic adsorption. The former is based on the interactions of proteins, peptides, and nucleic acids with short nonpolar chains on a support. This was first described in 1972 [113,114] following work that examined the role of spacer arms on the nonspecific adsorption of affinity columns [114]. Thiophilic adsorption, also known as covalent or chemisorption chromatography, makes use of immobilized thiol groups for solute retention [115]. Applications of this method include the analysis of sulfhydryl-containing peptides or proteins and mercurated polynucleotides [116]. 

Potuzak, H. and Dean, P.D.G., Affinity chromatography on columns containing nucleic acids, FEES Lett., 88, 161-166, 1978. 

Macromolecules such as proteins, polysaccharides, nucleic acids differ only in their physicochemical properties within the individual groups and their isolation on the basis of these differences is therefore difficult and time consuming. Considerable decreases may occur during their isolation procedure due to denaturation, cleavage, enz3rmatic hydrolysis, etc. The ability to bind other molecules reversibly is one of the most important properties of these molecules. The formation of specific and reversible complexes of biological macromolecules can serve as basis of their separation, purification and analysis by the affinity chromatography [6]. 

Enz)mies, inhibitors, cofactors, nucleic acids, hormones or cell chromatography can also be utilized as ligands in bioaffinity chromatography types. Examples of these methods include Receptor Affinity Chromatography and DNA Affinity Chromatography [21]. 

Affinity chromatography can be applied to the isolation and purification of virtually all biological macromolecules. It has been used to purify nucleic acids, enzymes, transport proteins, antibodies, hormone receptor proteins, drug-binding proteins, neurotransmitter proteins, and many others. 

Affinity chromatography Has a wide number of uses and can be applied to the isolation and purification of virtually all biomolecules. Specific applications include nucleic acid purification, protein purification from cell and tissues extracts, and antibody purification from blood serum. There are a number of matrices used for the construct, and some examples of these and their uses are as follows heparin columns to separate cholesterol lipoproteins, lectin columns to separate carbohydrate groups, and phenyl boronate columns to separate glycated haemoglobins. 

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