Chapter 3-Contd. Western blotting & SDSPAGE Western Blot Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.
Western Blot Proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are transferred to a sheet of special blotting paper called nitrocellulose. The proteins retain the same pattern of separation they had on the gel. ..Western Blot
The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which
cannot be seen at this time. ..Western Blot The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.
..Western Blot 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-aminoacid
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. ..SDS 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 Small molecules move through the polyacrylamide forest faster than big
molecules. Big molecules stays near the well. SDS-PAGE The end result of SDS- PAGE has two important features: 1) all proteins contain only primary structure & 2) all proteins have a large negative charge
which means they will all migrate towards the positive pole when placed in an electric field. The actual bands are equal in size, but the proteins within each band are of different sizes. Sample of SDS- PAGE
Protein gel (SDS-PAGE) that has been stained with Coomassie Blue. What happens after electrophoresis? 1. Fix the proteins in the gel and them stain them.
2. Electrophorectic transfer to a membrane and then probe with antibodies- (Western blotting) (Refer Western Blot first few slides) ..Western blotting Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein.
This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. This antibody is used as a probe to detect the protein of interest. Western Blot followed by SDS Proteins are separated using SDS-polyacrylamide gel electrophoresis which separates proteins by size.
Nitrocellulose membrane is placed on the gel. The actual blotting process may be active (electroblotting) or passive (capillary). Electroblotter is used for faster and more efficient transfer of protein from gel to membrane Sandwich of filter paper, gel, membrane and more filter paper is prepared in a cassette, which is placed between platinum electrodes. An electric current is passed through the gel causing the
proteins to electrophorese out of the gel and onto the nitorcellulose membrane. http://video.search.yahoo.com/search/vide o;_ylt=A0geuo8BkoVMBB4BustXNyoA?ei =UTF-8&p=SDS%20page&fr2=tab-web&fr =yfp-t-701 Terminologies..
The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. The northern blot is a technique used in molecular biology research to study gene expression by detection
of RNA. Southwestern blotting, based along the lines of Southern blotting (which was created by Edwin Southern) and first described by B. Bowen and colleagues in 1980, is a lab technique which involves identifying and characterizing DNA-binding proteins (proteins that bind to DNA). Terminologies..
Dot blot a mixture containing the molecule to be detected is applied directly on a membrane as a dot. Protein detection using the dot blot protocol is similar to western blotting in that both methods allow for the identification and analysis of proteins of interest.
Assignments 2-WAY-LEARNING on electro blotting- mages/video/ppt Western blot-PPT Northern blot-PPT Southern blot-PPT Dot Blot-PPT 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
A modul a fogaskerekek legjellemzőbb adata, mert a fogaskerék valamennyi méretét a modullal fejezzük ki. Az osztókör kerülete K = d * π, de kifejezhető a fogszám és az osztás által is K = z * p. Az elemi fogazású...
The physical optics code provides full 3-D simulation of the interaction of a propagating wavefront with the hot, thermally deformed crystal, without using parabolic approximation. For this purpose the code propagates the wave front in small steps through crystal and...
Here B is vertex. The two rays are called the arms of the angle. Here BA and BC are arms of angle ABC. TYPES OF ANGLES There are five types of angles:- Acute angle Right angle Obtuse angle Straight angle...
Kagan Cooperative Learning, 2009 La interdependencia positiva crea apoyo mutuo y confianza. Actividades para armar y reafirmar equipos están diseñados para ayudar a que los alumnos se conozcan, se acepten y se sientan incluidos. Kagan Cooperative Learning, 2009 Los alumnos...
Three Skeleton Key by George Toudouze Literary Focus: Suspense: anxious curiosity that hooks the readers' interest Foreshadowing: writers drop clues that hint at what may happen later; this intensifies suspense George Toudouze 1847- 1904 Born in France Playwright, essayist, and...
The Critical 5. The Critical Five was established in 2012 to enhance information sharing and work on issues of mutual interest between Australia, Canada, New Zealand, the United Kingdom and the United States.
Ready to download the document? Go ahead and hit continue!