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Chapter 3-Contd.��Western blotting & SDS-PAGE

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Western Blot

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  • 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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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

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  • 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

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