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Biochemical Methods Ii Notes

Pharmacology Notes > BIOL10212 Biochemistry Notes

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Biochemistry - Lecture 12 (08/03/2018)
Biochemical Methods II
PAGE (Polyacrylamide gel electrophoresis)

PAGE uses an electric field to move charged particles through a gel with pores.

Electrophoresis separates the charged molecules by their size as the larger molecules tend to move more slowly down the gel.

In order for PAGE to work all of the molecules have to be negatively charged.

Sodium Dodecyl Sulphate (SDS-page):

SDS is a large salt, of a long aliphatic acid [CH3-(CH2)11-SO4- Na+].
It is a detergent molecule with a dual character, as it is ionic at its head and hydrophobic on the chain.

At a neutral pH, SDS forms complexes (micelles) with polypeptide chains in their denatured form.
The approximately constant ratio for globular proteins typically is 1.4g SDS/ g of protein
(This can go up to 1.5-2 g as there is more SDS for membrane proteins).
e complexes are negatively charged and have a uniform shape, and the length of the micelle is proportional to the molecular weight of the protein.




SDS treatment ensures proteins are separated on the basis of their size by denaturing them and adding a uniform charge.
So therefore, SDS-PAGE can be used to estimate the mass of a protein.

Protein Purification:

Protein purification is the procedure by which you begin with a mixture of many proteins and end up with just one protein separated from the rest, allowing you to monitor the evolution of a system.

The specific activity relates to how much of the desired protein is present in the sample,
the higher the specific activity, the purer the sample.
To make the sample purer fractionation and chromatography is repeatedly carried out.


It is possible to monitor each of the purification stages if you load a sample into the gel after each cycle to display fewer and fewer bands, showing that less proteins are present.

The bands of the desired proteins will become thicker and thicker,
displaying that the sample is becoming purer. pI = The Isoelectric Point.


This form of protein separation exploits both the proteins mass and its isoelectric point.
The isoelectric point is the point at which the molecule (protein) has a net charge of 0 and this is dependent upon the pH and different proteins have different isoelectric points.

When you have a protein, and put it into a low pH environment, it will generally have a positive charge, and when at a high pH, a negative charge.
There is point on the pH scale where the net charge becomes 0, and this is the Isoelectric point.

Separation of proteins by Isoelectrofocusing


Isoelectrofocusing the protein sample involves placing it in the middle of the gel that is supplied with an electric field.
The proteins have not been modified and so retain their original charge, causing them to move towards either the positive or the negative electrode.
The gel is modified chemically so that it has different pH's from left to right, and so has a pH
gradient. Therefore, the proteins will move through different pH's and will reach the point where the pH is the same at the isoelectric point.
At the isoelectric point the charge will become zero and the movement will stop, called focusing, and the position of the protein becomes fixed.

2D Gel Electrophoresis:

From the electrofocusing experiment proteins are separated depending upon their isoelectric point.

However, it is possible for many proteins to share the same isoelectric point.

Therefore, you can further separate the proteins by rotating the gel 90° and treating it with SDS.
This allows a separation based on molecular weight to be carried out.

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