PAGE Electrophoresis…

Hafza Iftikhar

Basma Sarfraz

Komal Jameem

-Introduction

-Process technique-Uses and future Prospect

Electrophoresis is a laboratory technique for

separating molecules based on their charge

Electrophoresis…

The gel (matrix)

The gel (matrix) itself is composed of either agarose or polyacrylamide.

Polyacrylamide is a cross-linked polymer of acrylamide.

Polyacrylamide gels…

Have smaller pores than agarose, therefore high degree of resolving power.

Can separate DNA fragments which range in size from 10-500 bp.

DNA fragments which differ in size by one nucleotide can be separated from each other.

Polyacrylamide gel electrophoresis is mostly used to separate protein molecules.

Protein Electrophoresis

Proteins, unlike DNA, do not have a constant size to charge ratioIn an electric field, some will move to

the positive and some to the negative pole, and some will not move because they are neutral.

SDS-PAGE (PolyAcrylamide Gel Electrophoresis)…

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology:

to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight).

to separate proteins according to their size, and no other physical feature.

SDS…

SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges thus overwhelming positive charges in the protein.

The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein.

Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be solubalized by the detergent and all the proteins will be covered with many negative charges.

PAGE…

If the proteins are denatured and put into an electric field (only), they will all move towards the positive pole at the same rate, with no separation by size.

However, if the proteins are put into an environment that will allow different sized proteins to move at different rates.

The environment is polyacrylamide.

the entire process is called polyacrylamide gel electrophoresis (PAGE).

Page electrophoresis

techniques…

Basma Sarfraz

Sampling…

Samples may be any material containing proteins

Solid tissues:

Broken down mechanically using a

1. Blender (for larger sample volumes)

2. Homogenizer (smaller volumes)

3. Sonicator

4. Cycling of high pressure.

Soft tissues or cells:

Broken down by a combination of biochemical and mechanical techniques as

1. Filtration

2. Centrifugation

3. Electrophoresis

Materials Required For PAGE: Protein samples .

Molecular weight markers.

Acrylamide solutions (for resolving & stacking gels).

Isopropanol / distilled water .

Gel loading buffer.

Running buffer.

Staining, destaining solutions.

The equipment and supplies necessary for conducting SDS-PAGE includes: An electrophoresis chamber and power supply.

Glass plates(a short and a top plate).

Casting frame .

Casting stand.

Combs .

Preparing acrylamide gels…

Gels typically consist of

1. Acrylamide

2. Bisacrylamide

3. SDS

4. Buffer with an adjusted pH

5. Ammonium persulfate

6. TEMED

Recipe for a traditional 10% mini gel for SDS-PAGE:1. 7.5mL 40% acrylamide solution2. 3.9mL 1% bisacrylamide solution3. 7.5mL 1.5 M Tris•HCl, pH 8.74. Add water to 30mL5. 0.3mL 10% APS6. 0.3mL 10% SDS7. 0.03mL TEMED

It possesses several electrophoretically desirable features that make it a versatile medium. It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, can also withstand high voltage gradients, is amenable to various staining and destaining procedures, and can be digested to extract separated fractions or dried for autoradiography and permanent recording and can be prepared with a wide range of average pore sizes.

Sample preparation by using SDS, anionic detergent (HCl), tracking dye

(bromophenol blue)…

Preparation of gel solutions…

Stacking and Resolving gel’s role… Regardless of the system, preparation requires casting two different layers of acrylamide between glass plates.

The upper layer (stacking gel) includes the sample wells. It is designed to sweep up proteins in a sample between two moving boundaries so that they are compressed (stacked) into micrometer thin layers when they reach the separating gel.

The lower layer (separating, or resolving, gel) is responsible for actually separating polypeptides by size.

The ionic strength discontinuity between the loose stacking gel and the hard running gel leads to a voltage discontinuity as current is applied. The goal of these gels is to maximize resolution of protein molecules by reducing and concentrating the sample to an ultrathin zone (1-100 nm) at the stacking gel:running gel boundary. The protein sample is applied in a well within the stacking gel

The protein sample contains glycerol or sucrose so that it can be overlaid with a running buffer. Resolving gel 10% Stacking gel 5%

dH20 4.0 ml 5.65ml30% acrylamide mix 3.3 ml 1.65ml1.5M Tris pH8.8 2.5 ml pH 6.810% SDS 0.1 ml10% ammonium persulfate 0.1 mlTEMED 0.004ml

Solution pouring and gel preparation…

Buffers…

unreactive and not modify with most proteins.

Significance: Stabilizes the pH value to the desired value

Electrophoretic mobility- ions

Common buffers in SDS-PAGE include Tris, Bis-Tris, or imidazole.

Gel loading buffer: To make 10 mL of 4X stock: 2.0 ml 1M Tris-HCl pH 6.8.0.8 g SDS.4.0 ml 100% glycerol.0.4 ml 14.7 M β-mercaptoethanol.1.0 ml 0.5 M EDTA.8 mg bromophenol Blue.

Buffers preparation and their pouring…

Electroporesis separation depends upon:1. Protein’s size ( molecular weight)

2. Gel pores (density)

3. Voltage applied

Exceptions:

Glycoproteins

• Finding of approximate molecular weight…

By running molecular weight size markers of known molecular weight in a separate lane in the gel.

Further processes after electrophoresis… Staining:

 The gel may be stained most commonly with Coomassie Brilliant Blue R-250 or Silver stain.

Allowing visualization of the separated proteins, or processed further.

Result:

 Different proteins will appear as distinct bands within the gel. Alternatively, if the proteins are radioactive, the protein bands can be detected by autoradiography.

Stained gels can be dried and preserved, photographed or scanned with a recording densitometer to measure the intensity of the color in each protein band.

 different proteins will appear as distinct bands

within the gel

Western blotting

Weigh 0.25g of Coomassie Brilliant Blue R250 in a beaker. Add 90 ml methanol:water (1:1 v/v) and 10ml of acetic acid.

Flaws behind technique…

SDS-PAGE is usually the first choice as an assay of protein purity due to its reliability and ease.

1. Presence of SDS

2. Denaturing step

3. Aberrant migration of some proteins

4. Differently staining interferes with quantification.

preparative technique for the

purification of proteins-QPNC-

PAGE

Uses and Future prospective of PAGE…

komal jamim shehzadi

WHAT IS SPECIAL ABOUT SDS PAGE

SDS is a negatively charged detergent

Disrupt secondary and tertiary structures by breaking hydrogen bonds and unfolding proteins.

MASKS charge on protein so that all protein act the same as regards charge.

Prevents protein aggregation.

Prevents protein shape from influencing gel run.

Sodium Dodecylsulphate

Now we are ready to focus on the second half - PAGE.

SDS and Proteins

SDS

Protein

SDS and Proteins

So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains

In the presence of SDS all proteins have uniform shape and charge per unit length

SDS nonpolar chains arrange themselves on proteins and destroy secondary tertiary and quarternary structrure

Uses of SDS page…

Determine protein size Identify protein Determine sample purity Identify existence of disulphide bond Quantify amounts of protein Recycle back to the PM Recycle back to the TGN Western transfer always begin with SDS page

Cont…

Antibodies can be produced from protein excised from gel.

Sometimes can reconstitute proteins Mass spectrometry and proteomic studies Measuring Molecular Weight with SDS-PAGE Peptide mapping Protein concentration determination Protein crystallization

SDS-PAGE analysis of M. leprae protein antigens reacting with antibodies

from sera from lepromatous patients and infected armadillos.

A K Chakrabarty, M A Maire, and P H Lambert

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This article has been cited by other articles in PMC.

Abstract

Studies have been conducted to characterize M. leprae bacilli derived from infected armadillos. First, the proteins of the mycobacterial extracts were fractionated by SDS-PAGE. Subsequently, the proteins in the gel were electrophoretically transferred on a strip of nitrocellulose paper by the technique of 'electrophoretic blotting'. The separated bacterial protein bands, thus immobilized on the nitrocellulose paper were made to react immunologically with sera from the lepromatous patients, infected armadillo sera and other experimental mycobacterial antisera. It was observed that a majority of M. leprae proteins contained antigenic determinants also present on proteins of BCG. In addition, only two specific antigen bands of 33KD and 12KD were conspicuously detected by the patients' sera and the infected armadillo sera. These substances were further identified as polysaccharides or glycoproteins since they could only be stained by Schiff's reagent or alcian blue. Only 12KD glycoprotein band reacted with concanavalin A, whereas wheat germ agglutinin (WGA) did not show any reaction with them. These 33KD and 12KD glycoprotein antigens were found to lose their antigenicity after pepsin treatment and can be considered as glycoproteins. Further, radiolabelling experiments showed that 12KD antigen underwent radioiodination under usual conditions, but 33KD glycoprotein failed to be similarly radiolabelled. It is suggested that these protein antigens have M. leprae specific determinants on a cross-reacting component.