Affinity Chromatography

 Certain proteins bind strongly to specific small molecules. One can take advantage of this by developing an affinity chromatography system where the small molecule (ligand) is bound to an insoluble support. When a crude mixture of proteins containing the protein of interest is passed through the column, the ligand binds the protein to the matrix, whilst all other proteins pass through the column. The bound protein can then be eluted from the column by changing the pH, increasing salt strength or passing through a high concentration of unbound free ligand. For example, glutathione-S-transferase ( GST) is a protein with high affinity for the tripeptide glutathione (Figure 1). The technique is most frequently used with protein fusion constructs, but also has applications for a wide variety of other targets, including nucleotides, nucleic acids, immunoglobulins, membrane receptors, and even whole cells and cell fragments. Beaded agarose resins with reactive groups ( activated agarose ) for conjugation allow immobilisation of suitable small and macro-molecules. Most frequently used groups for conjugation include N -hydroxysuccinimide ( NHS) or cyanogen bromide (CNBr) for amino groups, and carbodiimides for carboxyl groups.

Fig1. Example of an affinity chromatography purification procedure. Absorbance at 280 nm is indicated by the blue line, conductivity by the dashed line and salt concentration (NaCl) by the grey dotted line. The red ticks indicate different fractions isolated, the contents of which are analysed on SDS-PAGE (shown at the bottom). A: Sample injection; due to the high protein concentration, the absorbance reaches the maximum recordable value; most protein passes through the column, unbound. B: Contaminating proteins eluting by way of a salt gradient. C: Elution of the fusion protein by injection of reduced glutathione.

Ion Exchange Chromatography

Proteins differ from one another in the proportions of the charged amino acids (aspartic and glutamic acids, lysine, arginine and histidine) that they contain.

Hence, different proteins possess different net charges at a particular pH. This difference is exploited in ion exchange chromatography, where the protein of interest is adsorbed to a solid support material bearing charged groups of the opposite sign (ion exchange resin). Proteins with the same charge as the resin pass through the column to waste; subsequently, bound proteins are selectively released from the column by gradually increasing the concentration of salt ions in the buffer passing through the column. These ions compete with the protein for binding to the resin, the more weakly charged protein being eluted at the lower salt concentration and the more strongly charged protein being eluted at higher salt concentrations. Alternatively, one may keep the salt concentration constant, but gradually change the pH of the eluting buffer.

Ion exchange chromatography can be used as an initial purification step, or as a ‘polishing’ step after a certain purity has been achieved because it is a relatively gentle purification method.

Frequently, fairly high purity of the target protein can be achieved with one simple gradient elution of an ion exchange column ( Figure 2), but often it may also be necessary to perform scouting experiments to determine the optimal run conditions involving buffer composition, length of gradient and type of media (anionic or cat ionic exchanger).

Fig2. Illustration of ion exchange chromatography. The top panel shows the chromatogram from a purification of a GST-fusion protein. Although the initial affinity purification step with glutathione was relatively pure, there are contaminating bands at ~28, ~66 and ~75 kDa. A column packed with a strong cation exchanger (MonoS ® ) was used to bind the target protein and a gradient of 0–100% 1 M NaCl in HEPES (pH 8.0) was applied using an ÄKTA HPLC system. Analysis of the isolated fractions show highly pure target protein in fractions 11–13, as judged by Coomassie Brilliant Blue staining.

Size-Exclusion Chromatography ( SEC)

Size differences between proteins can be exploited in molecular exclusion chromatography (also known as gel filtration). The size-exclusion medium consists of a range of beads with slightly differing amounts of cross-linking and therefore slightly different pore sizes. The separation process depends on the different abilities of the various proteins to adsorb to some, all or none of the cavities on the beads, which relates the retention time on the resin to the size of the protein. The method has limited resolving power, but can be used to obtain a separation between large and small protein molecules and therefore be useful when the protein of interest is either particularly large or particularly small. A major advantage is that this method is very gentle on proteins and is often used as a final stage in preparations destined for protein crystallography and other applications that require functional protein. Size-exclusion chromatography can also be used to determine the relative molecular mass of a protein. Furthermore, owing to the large size difference between inorganic ions and proteins, it is frequently used for desalting of protein solutions.

An important parameter to consider for size-exclusion chromatography is the con centration of the analyte; it should be as high as practicably possible. The higher the concentration the better the resolution due to reduced diffusion. Large proteins or protein complexes will pass through the medium and elute first, therefore it is very important to thoroughly equilibrate the column before use. Some amount of material will be lost in a column through dilution and surface interactions, therefore it is important to select the appropriate column size.

Hydrophobicity Interaction Chromatography (HIC)

Proteins differ in the amount of hydrophobic amino acids that are present on their surfaces. This difference can be exploited in salt fractionation, but can also be used in a higher-resolution method using hydrophobic interaction chromatography ( HIC). Typical resins include Butyl- and Phenyl-Sepharose, where butyl or phenyl groups are bonded to the agarose support matrix. The protein mixture is loaded on the column typically under high salt conditions (‘salting out’), where due to the hydrophobic effect interactions will occur between the immobilised hydrophobic groups on the resin and hydrophobic regions on the proteins. Proteins are then eluted by applying a decreasing salt gradient to the column and should emerge from the column in order of increasing hydrophobicity. However, some highly hydrophobic proteins may not be eluted, even in the total absence of salt. In this case, it is necessary to add a small amount of water-miscible organic solvent, such as propanol or ethylene glycol to the column buffer solution. This will compete with the proteins for binding to the hydrophobic matrix and will elute any remaining proteins. With individual proteins, it may also be possible to employ a triggered conformational switch to change between two different states of hydrophobicity (for example, the calcium-myristoyl switch observed in Visinin-like proteins), thus eliminating the requirement for high salt concentrations.

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الأسباب الرئيسية لنقص الطاقة في الجسم

الكاكاو وتأثيره على العين.. نتائج واعدة من تجارب مخبرية

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جيتكس إفريقيا.. المغرب يجمع "رواد التكنولوجيا" من 103 دول

طاقم أرتميس 2 يحطم رقما قياسيا ويستعرض الجانب البعيد للقمر

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مواضيع ذات صلة

Other Methods of Protein Purification

Producing Recombinant Protein

Engineering Proteins for Purification

حوامض أمينية محورة Modified Amino Acids

Amplification methods

Immunoelectrophoresis

Immunoprecipitation

Antibody production

Introduction to Immunological methods

Isoelectric focusing

Pore gradient gel electrophoresis

SDS-PAGE