Question

Proteins may be separated according to size by A. isoclectric focusing. B. polyacrylamide gel electrophoresis. C. ion exchange chromatography. D. molecular exclusion chromatography. E. reverse-phase HPLC.

Short answer

Answer: Molecular exclusion chromatography.

Step-by-step solution

Understand the methods

Each of the methods given in the options has its own purpose and separates proteins based on different properties. Here is a brief overview of the techniques: A. Isoelectric focusing: This method separates proteins based on their isoelectric points (pI). B. Polyacrylamide gel electrophoresis (PAGE): In this method, proteins are separated based on their size, shape, and charge. C. Ion exchange chromatography: This method separates proteins based on their differences in charge by using a charged stationary phase. D. Molecular exclusion chromatography (also known as size-exclusion chromatography, or SEC): This method separates proteins based on their size. E. Reverse-phase HPLC: This method separates proteins based on their hydrophobicity, using a non-polar stationary phase.

Choose the correct answer

Based on the descriptions of the methods, the technique used for separating proteins according to size is the molecular exclusion chromatography (size-exclusion chromatography). Therefore, the correct answer is: D. Molecular exclusion chromatography.

Key concepts

Isoelectric Focusing

Isoelectric focusing (IEF) is a method used to separate proteins based on their isoelectric points (pI), which is the pH at which a protein has no net charge. Proteins are applied to a gel with a pH gradient and an electric field is applied. As proteins move through the gradient, they reach a point where their net charge is zero and they focus into sharp bands. This technique is valuable in analyzing protein mixtures with similar sizes but different pI values.

Understanding isoelectric focusing is crucial for students studying biochemistry or molecular biology. It’s particularly used before another type of analysis like polyacrylamide gel electrophoresis (PAGE), to further distinguish proteins that have similar molecular weights but different isoelectric points.

Polyacrylamide Gel Electrophoresis

Polyacrylamide Gel Electrophoresis (PAGE) is commonly used to separate proteins based on their size, shape, and charge. In PAGE, proteins are denatured with a detergent like SDS (sodium dodecyl sulfate) to ensure they have a uniform negative charge and linear shape. This neutralizes any differences in charge and shape, allowing the separation essentially by size as the proteins migrate through the polyacrylamide gel towards the positive electrode.

Students should understand the importance of the polyacrylamide matrix which acts like a sieve, letting smaller proteins travel faster than larger ones. SDS-PAGE is a specific type of PAGE that is especially popular because of its ability to separate proteins solely based on their molecular weight, making it a key technique in the analysis of protein samples.

Ion Exchange Chromatography

Ion exchange chromatography involves the separation of proteins based on their net charge. This technique uses a charged stationary phase, such as a column containing resin. Proteins with a charge opposite to that of the stationary phase will bind to the resin, while those with the same charge will pass through.

For students tackling this concept, it's vital to recognize the need for a gradient of salt concentration or pH in the mobile phase which is used to elute the proteins off the column. The strength of interaction between the protein and the resin, along with the protein's charge, determines the order in which proteins elute from the column.

Molecular Exclusion Chromatography

Molecular exclusion chromatography, also known as size-exclusion chromatography (SEC), is a technique that separates proteins based on their size. In this method, the stationary phase consists of beads with pores of a controlled size. Larger molecules cannot enter the pores and are eluted first, whereas smaller molecules enter the pores and are delayed, allowing for their separation.

Educators stress the utility of SEC for not only separating proteins by size but also for determining the molecular weight and quaternary structure. As this was the method needed for the original exercise question, students should be familiar with its application in protein purification processes.

Reverse-Phase HPLC

Reverse-phase High Performance Liquid Chromatography (HPLC) is a powerful technique used to separate proteins based on hydrophobicity. This method employs a non-polar stationary phase and a polar mobile phase. Proteins with greater hydrophobicity will bind more strongly to the non-polar stationary phase and will elute later than more hydrophilic proteins.

When learning about reverse-phase HPLC, students should focus on the role of gradient elution. This involves gradually increasing the concentration of a non-polar solvent in the mobile phase, which eventually causes the protein to elute from the column. Reverse-phase HPLC is useful for separating complex protein mixtures and is often used in conjunction with mass spectrometry for protein identification.