Determination Of Molecular Weight Of A Protein By Gel Filtration

3339 words - 13 pages

Introduction

Purification of proteins is an essential technique that provides biochemists with the ability to obtain a sample that contains a specific protein of interest. In order to purify a protein biochemists typically obtain the specific extract, precipitate the protein using ammonium sulfate, preform gel filtration, and lastly run ionic and affinity chromatography. Understanding the use and methods of the different chromatography techniques is of crucial importance in order to successfully purify and conclude the molecular weight of an unknown sample using elution volumes of molecular weight standards.
Gel filtration or exclusion chromatography is preformed to isolate proteins of different sizes between various phases, such as solid or gel and liquid phases. If solid, the gel and beads are denoted as sorbent. Sample mixtures have two phases that they move through, a stationary and mobile phase. The stationary phase is a solid or sorbent, while the mobile phase is gas or water. The process of movement of a sample down a column is referred to as development.
Gel exclusion chromatography is an ideal method used to isolate molecules that are sensitive to harsh environments such as changes in pH or varying concentrations of metal ions. When running gel exclusion chromatography, smaller biomolecules typically penetrate the gel more and thus, take more time to elute through the column. Furthermore, larger biomolecules are too large to penetrate the gel pores and elute through the column at a faster rate. Upon coming out of the Sephedex (or out of the column) biochemists collect fractions, to further the continuation of the experiment. The diagram depicted in the figure below illustrates large molecules and small molecules eluting and the larger biomolecules making their way out of the Sephadex quicker.


Figure 1: Gel filtration columns
Selfe, S

Gel exclusion chromatography has given biochemists the opportunity to learn more about specific proteins varying characteristics such as size, shape, and solubility. Biochemists are able to utilize different equations to solve for these characteristics. The elution volume (ve ) is the volume of solvent needed for a solute to elute down a column; this is the volume of the fraction corresponding to the highest absorbance. The void volume (vo) is the volume outside of the gel particles. In order to conclude the void volume of a column one would utilize the elution volume of Blue Dextran-2000 (molecular weight of 2.00 x 106 g/mol). Each fraction collected contains 1.00 mL of the sample. For example, to determine the volume of the 2nd fraction: Volume of sample in each fraction (1 mL) x tube number (2) = 2 mL

The total volume (vt) of a packed column is calculated using the following equation:
Vt = Vi + Vg + Vo

where Vi is the internal volume or the volume inside the gel particles , Vg is the volume due to the solid walls and cross-linked internal chains of gel particles, and Vo...

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