Question

In: Chemistry

1. How critical is it to have a small sample volume to load on the following...

1. How critical is it to have a small sample volume to load on the following kinds of chromatography in order to get good resolution? Size Exclusion, ion exchange, affinity chromatography. Consider how the proteins interact with the column material. This experiment uses an 80% ammonium sulfate cut, however, as you look at the ammonium sulfate concentration table, other percentages are indicated.  Why do you think 80% was selected in this case? What is another way that you could use ammonium sulfate precipitation in a protein purification?

Solutions

Expert Solution

For Better resolution in size exlusion the volume should be based on below principle.

Ideally, proteins are separated based on size, limiting the resolution between analytes. In instances of column overloading, resolution between the analytes can deteriorate. Ideal volume loads correspond to sample volumes between 5–10% of the total column volume.

Ion exchange

Sample loads can be increased if resolution is satisfactory or when using a step elution ,For good resolution use around 20% of column capacity.

Resolution is more readily affected by changes in flow rate or sample load due to the intermediate forms of charge interaction which occur this is incase of weak exahngers.

Affinity Chromatography,

Affinity chromatography is very selective and provides high resolution with an intermediate to high sample loading capacity. The protein of interest is tightly bound to the resin under conditions that favor specific binding to the ligand, and unbound contaminants are washed off. The bound protein is then recovered in a highly purified form by changing conditions to favor elution. Elution conditions may be specific, such as a competitive ligand, or nonspecific, such as changing pH, ionic strength, or polarity. The target protein is eluted in a purified and concentrated form.

COLUMN VS PROTIEN INTRACTION AND HOW CAN WE PREVENT.

Affinity chromatography is based on specific interactions between proteins and column ligands, salts, which weaken ionic interactions, may enhance other types of interactions. Thus, salts may be ineffective in eluting proteins from the affinity resin.

The salts modulate affinity of the proteins for particular columns and nonspecific protein–protein or protein–surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein–protein or protein–surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers.

why Use of 80% Ammonium sulfate.

Usually at 80% saturation all proteins will be precipitated.

The main logic behind salt precipitation is to nullify charge on protein molecule essential of its solubility. Using salt like ammonium sulphate led saturation and precipitation of protein. The concentration of salt required may vary from protein to protein so its a matter of few trails to have good results.


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