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)

Explain procedure for checking the presence of rhodamine b, Explain Procedu...

Explain Procedure for Checking the Presence of Rhodamine B? Conduct the experiment following the procedure given herewith: 1. Pour liquid paraffin or mineral oil in a Petri

SCINCE, I WANT JOB FOR TEACHING SCINCE SUBJECT FOR ONLINE

I WANT JOB FOR TEACHING SCINCE SUBJECT FOR ONLINE

Explain empiric initial therapy, Empiric Initial Therapy  Until suscept...

Empiric Initial Therapy  Until susceptibility results are available, empiric initial treatment consists of a 4-drug regimen of isoniazid, rifampin, pyrazinamide and ethambutol.

Exon, Exon are those parts of a genomic DNA sequence which will be represe...

Exon are those parts of a genomic DNA sequence which will be represented in final, mature mRNA t A contiguous segment of the genomic DNA which codes for a polypeptide in the gene.

Define the magnesium requirements in body, Define the Magnesium Requirement...

Define the Magnesium Requirements in body? Let us learn about the magnesium requirements. Since plant foods are particularly high in magnesium, on a vegetarian diet with plenty

Define the effect of scfa on sodium absorption, Define the effect of SCFA o...

Define the effect of SCFA on Sodium Absorption? SCFAs (short chain fatty acids) have a stimulatory effect on sodium absorption from colonic lumen. The unionized SCFA crosses th

What property of water helps, South Dakota and Oregon are at similar latitu...

South Dakota and Oregon are at similar latitudes, and yet, on average, Oregon has warmer winters and cooler summers than South Dakota does. What property of water helps account for

What are good pharmacovigilance process, Question 1: What are Good Phar...

Question 1: What are Good Pharmacovigilance Process and Good Post-Marketing Surveillance Process? How do they differ from each other? Define and illustrate briefly Good Post

What are the positions of myosin and actin molecules, Q. What are the posit...

Q. What are the positions of myosin and actin molecules in the sarcomere before and during the muscle contraction? Schematically actin filaments attached perpendicularly to bot

Filaments of a sarcomere in a skeletal muscle is decreasing, When the overl...

When the overlap between the thin and thick filaments of a sarcomere in a skeletal muscle is decreasing, the A. total length of the I band minus the length of the H zone is dec

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