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Chapter 33: Problem 2

Consider the requirements for sample application in PAGE and IEF. Explain why in PAGE the sample is applied in a discrete narrow band, usually at the cathodic end of the gel, while in IEF the sample can be applied at any point along the length of the gel without concern about location of narrowness of the sample zone.

Short Answer

Expert verified
In PAGE, samples are applied in a narrow band at the cathodic end as separation is based on protein size, hence a narrower starting position ensures good resolution. In IEF, sample application location isn't critical because proteins will migrate and reach a point of zero net charge (their pI), regardless the initial location.

Step by step solution

01

Understanding the Basics

PAGE and IEF are methods used for protein separation based on their physical and chemical properties. In PAGE, samples are usually applied in a discrete narrow band at the cathodic end, while in IEF, the sample is applied along the length of the gel.
02

PAGE Procedure

In PAGE, the samples need to be applied in a narrow band at the cathodic end because the separation is solely based on the size of the proteins. The proteins migrate towards the anode due to their negative charge in the presence of a detergent (SDS). Since all proteins have similar charge to mass ratio, making the starting sample be as discrete and narrow as possible ensures good resolution.
03

IEF Procedure

In IEF, the separation is based on the isoelectric point (pI) of the proteins, which is the pH at which proteins have no net charge. In IEF, a pH gradient is established and proteins move until they reach a point where their net charge is zero. Because of this, the exact point of application isn't critical as proteins will migrate to their pI regardless of where they started.

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Most popular questions from this chapter

Find out why the net charge on a protein molecule varies with pH. Identify the amino acids primarily responsible for determining the net charge on a protein molecule and draw simple diagrams to represent the ionisation of their side chains, indicating how you would expect these side chains to be charged at acid, neutral and alkaline \(\mathrm{pH}\) values.
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