Gel electroporosis

SDS GEL ELECTROPHORESIS AND BLOTTING TECHNIQUES.

ByY.PRATHAP

M.Pharm I- Sem (PHARMACEUTICS).

Roll no: 256213886031.

Under the guidance of: Asst.Prof . Mr. Uttam Prasad Panigrahi .

M.PHARM,(PAT).

CONTENTS:SDS Gel electrophoresis. Introduction. Principle. Instrumentation. Process. Applications.

Blotting techniques. Types & applications. References.

INTRODUCTION OF 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-PAGE

SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it.

Fig.1Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing negative and positive charges due to the charged R-groups in the protein. The large H's represent hydrophobic domains where nonpolar R-groups have collected in an attempt to get away from the polar water that surrounds the protein. After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative charges which overwhelms any positive charges the protein had due to positively charged R-groups. The resulting protein has been denatured by SDS (reduced to its primary structure-amino acid sequence) and as a result has been linearized.

..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

Small molecules move through the polyacrylamide gel faster than big molecules.

Big molecules stays near the well.

PRINCIPLE: PAGE (Polyacrylamide Gel Electrophoresis), is an

analytical method used to separate components of a protein mixture based on their size. The technique is based upon the principle that a charged molecule will migrate in an electric field towards an electrode with opposite sign.

The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size. After the visualization by a staining (protein-specific) technique.

INSTRUMENTATION

APPARATUS: Apparatus for gel electrophoresis are relatively simple .

Electrophoresis cells are essentially plastic boxes with anode and cathode compartments. Electrodes(usually platinum wire) and jacks for making electrical contact with the electrodes.

Gels are held vertically between the electrode chambers during the run. Gel cassettes have open tops and bottoms. The bottom is sealed with a gasket during gel formation and the top is open to receive monomer solution. The top and bottom ends are open and in contact with buffer for electrophoresis.

High voltage direct current supplies provide electrical power for electrophoresis.

Micropipettes, test tubes and heating blocks are the sample handling necessities.

PARTS OF THE SYSTEM Gel support medium Agarose. Polyacrylamide(PA). Detergent: sodium dodecyl sulfate(SDS). Buffer : the electrical current in an electrophoresis cell is

carried largely by the ions supplied by the buffer compounds. Proteins constitute only a small portion of the current carrying ions in an electrophoresis cell. So buffers supply current carrying ions, maintain desired PH, provide a medium for heat for dissipation . Ex : Tris-acetate-EDTA and Tris- borate -EDTA.

DC Power supply.

PROCESS:

Sample preparation.

Prepare 2X sample buffer consisting of 0.5M Tris-HCl, pH 6.8, 4.4% SDS, 300mMMercaptoethanol, 10mg/ml Bromophenol Blue and mix with equal volume of sample .Bring to 95° C for 10 minutes, cool to room temperature before loading. If particulate is present, centrifuge samples 5 minutes at 14k RPM in microcentrifuge, and load the gel.

ENTIRE PROCESS DIAGRAM OF SDS GEL ELECTROPHORESIS.

PROTEIN VISUALIZATION ON GELS

• Immediately after electrophoresis proteins in the gels are precipitated by either adding alcohol containing solutions or strong acids (e.g. TCA)

• DNA may be visualized using ethidium bromide.

Protein are often stained by Coomassie Brilliant Blue dye or by photography-like treatment with AgNO3 (silver staining)

There are many other stains available (e.g. Stains-all, fluorescence probes etc.)

 PROTEIN GEL (SDS-PAGE) THAT HAS BEEN STAINED WITH COOMASSIE BLUE.

EXAMPLE OF SILVER STAINED GEL

APPLICATIONS: SDS PAGE is a useful method for separating and

characterizing macromolecules like DNA, RNA and proteins.

In Forensic , DNA Fingerprinting: men proving or disproving paternity by this technique. Used as witness.

The human Genome project. Illness: It can help scientists to identify certain

damaged genes . It can also help to identify certain genetic diseases like sickle cell anemia, also identify viruses.

Blotting : Separation of restricted genomic DNA prior to southern blotting and RNA prior to northern blotting.

BLOTTING

TECHNIQUES.

WHAT IS BLOTTING?

Technique for transferring DNA RNA Proteins

onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.

TYPES OF BLOTTING TECHNIQUES

BLOTTING TECHNIQUES

Southern Blot

It is used to detect the DNA.

Northern blot

It is used to detect the RNA.

Western blot

It is used to detect proteins.

BLOTTING SHEET

Whatman 3mm paper….. world’s most widely used blotting paper.

REASON???? high quality purity consistency

(0.34 mm) is used extensively in electrophoresis for lifting of sequencing gels.

CREATING THE SANDWICH

The sandwich consists of :

filter paper 

Nitrocellulose membrane

gel matrix

another piece of filter paper

SOUTHERN BLOTTING

History: Sir Edwin Southern

Developed in 1975

SOUTHERN BLOTTING

“Used to detect the DNA” This method Involves: Separation Transfer Hybridization. This DNA can be: Single gene Part of a larger piece of DNA……..viral genome

“The key to this method is Hybridization”

HYBRIDIZATION

“Process of forming a dsDNA molecule between a ssDNA probe and a ss-target

patient DNA”

PRINCIPLE

The mixture of molecules is separated. Immobilized on a matrix. Probe addition to the matrix to bind to the molecules. Unbound probes are removed.

“The place where the probe is connected

corresponds to the location of the immobilized

target molecule.”

STEPS IN SOUTHERN BLOTTINGThe DNA is digested

Fragments

Gel electrophoresis

Transfer to membrane

Probing

Autoradiogram

APPLICATIONS

Southern blotting is used in: Gene discovery  Mapping Evolution Development studies Diagnostics Forensics

NORTHERN BLOTTINGHistory:

Northern blotting was developed by James Alwine and George Stark at Stanford University.

Northern blotting is a technique for detection of specific RNA sequences

STEPS INVOLVED IN N.B

RNA isolation

Loading of sample on Agarose gel

Blotting on nitrocellulose membrane

Labeling with probe

Washing to remove unbound probe

Detection by autoradiogram

APPLICATIONS

A standard for the direct study of gene expression at the level of mRNA (mRNA transcripts)

Detection of mRNA transcript size Study RNA degradation Study RNA splicing - can detect alternatively spliced

transcripts Study RNA half-life Study IRES (internal ribosomal entry site) – to remove

possibility of RNA digestion vs. 2nd cistron translation.

WESTERN BLOTTING

Discovery??? Dr. Douglas Lake of the University of Arizona

School of Medicine's Department of Microbiology and Immunology

“A technique in which proteins are separated by gel electrophoresis and transferred to a membrane sheet. A specific protein is then identified through its reaction with a labeled antibody.”

PRINCIPLE

This technique works on the principle on

“Antigen-Antibody” relationship

PREREQUISITE FOR W.B

•The SDS PAGE technique is a prerequisite for Western blotting.

“SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it.”

STEPS IN W.B1. Gel electrophoresis:

The proteins are separated according to size.

2. Membrane Transfer: Transferring to nitrocellulose by applying current.

3. Blocking: Done to prevent non-specific protein interactions between the membrane and the antibody protein.

APPLICATIONS

To identify the specific proteins

To identify their masses

The confirmatory HIV test to detect anti-HIV antibody in a human serum sample.

The definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').

Some forms of Lyme disease testing also employ Western blotting. 

REFERENCES: Introduction to Biotechnology by W.J. Thieman and

M.A. Palladino. Pearson & Benjamin Cummings 2nd edition.

http://www.toodoc.com/SDS-PAGE-ppt.html http://www.bio.davidson.edu/courses/genomics/

method/Westernblot.html http://en.wikipedia.org/wiki/Dot_blot Bio analytical techniques by M.L.Srivastava http://amrita.vlab.co.in/index.php. Wikipedia. www.authorstream.com.