Define polyacrylamide gel electrophoresis (page), Biology

Assignment Help:

Define Polyacrylamide gel electrophoresis (PAGE)?

Acrylamide gel has the advantage over starch in that it is easier to prepare and is more inert, the pore size can be varied in a controlled manner and over a wider range, and the technique is similar with a shorter running time. In PAGE, gels are made by the free radical induced polymerization of the acrylamide monomer with N,N' methylenebisacrylamide as cross linking agent; in the presence of an initiator N, N, N1, N1- tetramethyl ethylenediamine (TEMED) in a buffer of selected pH and ionic strength and in the presence of a catalyst such as ammonium per sulphate to initiate polymerization as soon as it is introduced into the monomer. Photochemical catalysis with riboflavin and a bright light is an alternative. The rate of polymerization is proportional to the TEMED concentration. Electrophoresis can be carried out in cylindrical glass tubes or by using vertical or horizontal flat-bed equipment. In the simplest form of PAGE, a tube 3 to 10 cms long in which the gel has been polymerized is suspended vertically between an upper and a lower buffer reservoir. The buffer, which is the same in both reservoirs and the gel, has a pH (about 9 for protein) such that the macromolecules have net negative charges and hence migrate to the anode in the lower reservoir. Each sample which can contain as little as 10 µg of macromolecular material is dissolved in a minimal amount of a relatively dense glycerol or sucrose solution to prevent it from mixing with the buffer in the upper reservoir and is applied to the top of the gel.

A direct current of 300 V is passed through the gel till the components are separated into a series of discrete bands (30-90 minutes), the gel is removed from its holder, and the bands are visualized by an appropriate method. Using this technique a protein mixture of 0.1 to 0.2 mg can be resolved into as many as 20 discrete bands. The very large pores needed for the PAGE of large molecular mass compounds (> 200 kDa) requires gels with very low polyacrylamide concentrations (< 2.5%) and hence they are too soft to be usable. This difficulty is overcome by using agarose alone or mixed with polyacrylamide. For example, a 0.8% agarose gel is used for the electrophoretic separation of nucleic acids with molecular masses of upto 50,000 KDa. The bands resulting from a gel electrophoretic separation can be located by a variety of techniques.


Related Discussions:- Define polyacrylamide gel electrophoresis (page)

Biosphere, Biosphere Biosphere is that part of the earth where life can...

Biosphere Biosphere is that part of the earth where life can exist. It is a narfqy layer around the surface of the earth. If you visualise the earth to be the size of an apple

Infusion pump, Infusion Pump As you'have learnt earlier, the amount o...

Infusion Pump As you'have learnt earlier, the amount of fluid to be administered to neonate is based on the neonate's weight and physiological status. It is recommended that

Describe the pulmonary artery, Q. Describe the pulmonary artery? The ma...

Q. Describe the pulmonary artery? The main pulmonary artery is seen on the PA view, as its left border forms the pulmonary bay. The right pulmonary artery runs horizontally to

Explain about blood pressure, Explain about Blood pressure Blood press...

Explain about Blood pressure Blood pressure is explained as the force exerted by the blood against the walls of the arteries. Blood pressure (BP) can be described as the fo

Describe the origin of muscle cramps and pains, Q. How can the knowledge ab...

Q. How can the knowledge about fermentation describe the origin of muscle cramps and pains after intense physical exertion? A typical fermentation process due to oxygen scarcit

History of cell biology, HISTORY OF CELL BIOLOGY Scientific knowledge gro...

HISTORY OF CELL BIOLOGY Scientific knowledge grows with the development of new tools and techniques for studying various physical and biological processes. This is true also of t

How many cc of basaljel in the medicine cup, The doctor ordered 3 teaspoons...

The doctor ordered 3 teaspoons of Basaljel suspension in water or juice. How many cc of Basaljel (aluminum carbonate) should the nurse place in the medicine cup?

Assessment of aplastic anaemia, Assessment   The patient will present w...

Assessment   The patient will present with striking pallor, irritability and lethargy due to anaemia, bledding or bruising and petechiae due to thrombocytopenia, fever and infe

Explain risk management, Explain Risk Management Risk Management :  ...

Explain Risk Management Risk Management :  The  process  of weighing  policy  alternatives  to accept, minimize or reduce assessed risks and  to select and  implement approp

Write Your Message!

Captcha
Free Assignment Quote

Assured A++ Grade

Get guaranteed satisfaction & time on delivery in every assignment order you paid with us! We ensure premium quality solution document along with free turntin report!

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd