page analysis of serum igg's

7/30/2019 PAGE Analysis of Serum IgG's

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Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis is used to determine the molecular weight of proteins, todetermine the purity of proteins, and to identify proteins by the use of immunoblotting (this

technique identifies proteins blotted onto nitrocellulose by the use of antibodies). It has been

used as a source of proteins for amino acid sequencing and also as a source of antigen for the

immunization of animals to obtain antibodies against the purified protein.

Polyacrylamide is the most commonly used matrix for protein electrophoresis. Polyacrylamide

gels are run vertically and are much thinner than agarose gels. The vertical gels are cast

between two plates and are connected to two different buffer tanks. Polymerization is sensitive

to oxygen thus casting between two plates reduces the influence of oxygen on the

polymerization process.

Buffer systems used in gel electrophoresis: Buffer systems in gel electrophoresis are divided

into two types; continuous buffers and discontinuous buffers. It is called a continuous buffer

system if the same buffer ions are present throughout the gel and if the pH of the gel remains

constant. When this type of buffer is used the protein sample is loaded directly in the gel that is

used to resolve the proteins into bands. The system that you used in agarose gel electrophoresis

had a continuous buffer.

In the discontinuous buffer system a second or stacking gel is placed on top of the resolving

gel; the proteins are loaded into the stacking gel (see Fig 4). In the Laemmli1 system, different

buffers are used in the stacking and resolving gels. Both the pH and the concentration of thecomponents are different. These buffers in turn are different from the tank buffer in both

concentration and in pH.

There are two particularly attractive features of the discontinuous buffer system. One is that a

dilute protein sample in the well is concentrated at the interface of the stacking and resolving

gels before entering the resolving gel (Fig 1). The second attractive feature is the ability of this

system to resolve proteins into readily distinguishable and relatively narrow protein bands that

are readily cut out. Once removed the protein in the band may be injected as an antigen or

microsequenced.

SDS -PAGE: Sodium dodecyl sulfate (SDS), also known as sodium laurel sulfate, is an anionic

detergent that binds to proteins. SDS has two important effects on proteins:

1. SDS disrupts non-covalent bonds in proteins resulting in their denaturation

2. SDS converts all proteins to negatively charged molecules. As a result all proteins

migrate towards the positively charged anode.

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These two effects permit one to separate proteins based only on their molecular weights.


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1 2 3 4 5 6 7 8 9 10

Fig 1. Schematic of basic setup for polyacrylamide gel electrophoresis. Lanes 1-3 show

samples shortly after loading into the wells. Lanes 4-6 show samples after the dye has

just entered the stacking gel. Lanes 7-9 show samples that have been resolved into their

component parts in the resolving gel. Lane 10 shows molecular weight markers that have

been run so that the molecular weights of the resolved components of the samples can be

determined. The largest protein is the uppermost band in any lane. The next largest

protein would be the second highest band in any lane and so forth. Note the different

buffers and the presence of two different gel that compose a discontinuous buffer gel.

Assembling the Vertical Gel Apparatus: Work in pairs (there may be one student working

alone if the class has an odd number of students). Assemble the vertical gel electrophoresis

unit, according to the demonstration given in class.

Cathodal Buffer Tank0.025M Tris, 0.192M

glycine, pH 8.3

Stacking Gel

0.125M Tris-HCl pH

Sample Wells

Resolving or

Runni

ng Gel

Anodal Buffer Tank0.025M Tris, 0.192M

glycine, pH 8.3

Stacking Proteins

Each BandRepresentsADifferent Molecular

Highest MW

Protein

Lowest MW

Protein


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Experimental results

page analysis of serum igg's

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