chapter 3-contd. western blotting & sds- page. western blot western blots allow investigators to...
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![Page 1: Chapter 3-Contd. Western blotting & SDS- PAGE. Western Blot Western blots allow investigators to determine the molecular weight of a protein and to measure](https://reader036.vdocuments.mx/reader036/viewer/2022062308/56649d795503460f94a5bfd2/html5/thumbnails/1.jpg)
Chapter 3-Contd.
Western blotting & SDS-PAGE
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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.
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…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.
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..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.
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..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.
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..Western Blot
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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.
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…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.
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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.
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..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.
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..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.
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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).
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..PAGE
• Small molecules move through the polyacrylamide forest faster than big molecules.
• Big molecules stays near the well.
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…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.
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The actual bands are equal in size, but the proteins within each band are of different
sizes.
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Sample of SDS- PAGE
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Protein gel (SDS-PAGE) that has been stained with Coomassie
Blue.
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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)
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..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.
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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.
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• http://video.search.yahoo.com/search/video;_ylt=A0geuo8BkoVMBB4BustXNyoA?ei=UTF-8&p=SDS%20page&fr2=tab-web&fr=yfp-t-701
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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).
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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.
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Assignments
2-WAY-LEARNING
• on electro blotting- mages/video/ppt
• Western blot-PPT
• Northern blot-PPT
• Southern blot-PPT
• Dot Blot-PPT
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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