Scribd Logo
Upload

Welcome to Scribd!

  • 19 views

    3 Western Blotting

    Uploaded by

    Uzair Ahmed
    Western blotting is a widely used technique to detect specific proteins in tissue or cell extracts. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. The key steps are lysing cells or tissue to extract proteins, separating proteins by molecular weight on a polyacrylamide gel, transferring to a membrane, blocking the membrane to prevent nonspecific antibody binding, incubating with primary and secondary antibodies, and detecting the target protein. This allows researchers to detect and analyze changes in protein expression and post-translational modifications.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd

    3 Western Blotting

    Uploaded by

    Uzair Ahmed
    19 views23 pages
    Western blotting is a widely used technique to detect specific proteins in tissue or cell extracts. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. The key steps are lysing cells or tissue to extract proteins, separating proteins by molecular weight on a polyacrylamide gel, transferring to a membrane, blocking the membrane to prevent nonspecific antibody binding, incubating with primary and secondary antibodies, and detecting the target protein. This allows researchers to detect and analyze changes in protein expression and post-translational modifications.

    Copyright

    © © All Rights Reserved

    Available Formats

    PPT, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Western blotting is a widely used technique to detect specific proteins in tissue or cell extracts. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. The key steps are lysing cells or tissue to extract proteins, separating proteins by molecular weight on a polyacrylamide gel, transferring to a membrane, blocking the membrane to prevent nonspecific antibody binding, incubating with primary and secondary antibodies, and detecting the target protein. This allows researchers to detect and analyze changes in protein expression and post-translational modifications.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    19 views23 pages

    3 Western Blotting

    Uploaded by

    Uzair Ahmed
    Western blotting is a widely used technique to detect specific proteins in tissue or cell extracts. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. The key steps are lysing cells or tissue to extract proteins, separating proteins by molecular weight on a polyacrylamide gel, transferring to a membrane, blocking the membrane to prevent nonspecific antibody binding, incubating with primary and secondary antibodies, and detecting the target protein. This allows researchers to detect and analyze changes in protein expression and post-translational modifications.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    You are on page 1of 23

    Western Blotting

    Dr. Usman Ali Ashfaq


    Dept. of Bioinformatics and Biotechnology, GC University,
    Faislabad

    Dept of Bioinformatics and Biotechnology, GC University, Faislabad


    Western Blot

    •The western blot is a widely


    accepted analytical technique used to
    detect specific proteins in the given sample
    of tissue, homogenate or extract

    • Method developed by George Stark

    • The name Western blot was given to the


    technique by W. Neal Burnett

    Dept of Bioinformatics and Biotechnology, GC University, Faislabad


    Western blotting
    • Detects proteins and estimates their
    molecular weight.
    • Detects changes in phosphorylation and
    lipid modifications.
    • Used to detect changes in protein
    expression.
    Protein Lysis Buffer

    • Buffer (Tris or Hepes buffer with pH 7-8)


    • Salt (usually NaCl 150mM (low) to
    500mM(high)
    • Chelating agent (EDTA or EGTA)
    • Detergent
    • Protease inhibitor
    • Phosphatase inhibitor (optional)

    Dept of Bioinformatics and Biotechnology, GC University, Faislabad


    Lysing Cells
    • Treat cells with appropriate conditions depending on
    the experiment
    • Pellet cells and lyse them in the appropriate lysis
    buffer.
    • Most important ingredient in lysis buffer is detergent.
    – Most stringent to weakest
    • SDS
    • NP-40
    • Triton X-100
    • Tween 20
    • Digitonin
    • CHAPS
    • Brig
    SDS covers proteins in a net negative charge
    Addition of 2-mercaptoethanol reduces disulphide bonds and
    Boiling is used to further denature proteins.

    Charged R groups
    +
    -
    -
    -
    + +
    H
    -

    +
    + Hydrophobic areas
    H
    +
    -
    -

    -
    - -
    Before SDS
    - - -
    -
    - - - -
    - - - -

    Migrate in gel according to mass


    • Second most important ingredient is protease
    inhibitors.
    – Once proteins are denatured by detergents, they are
    susceptible to degradation by proteases.
    – Need more than one inhibitor since there are lots of
    proteases.
    – Protease inhibitors
    • Aprotinin
    • Leupeptin
    • PMSF (phenylmethylsulfonyl fluoride)
    – Add immediately before lysis since PMSF activity decreases
    over time in aqueous solutions. About 15-30 minutes of activity.
    Phosphatase inhibitors
    • Need to add to lysis buffer if using
    phopshospecific antibodies or suspect protein of
    interest is phosphorylated
    • Inhibitors
    – ZnCl2
    – NaF
    – Na-Vanadate (tyrosine phosphatase inhibitor, add
    prior to lysis since only active in pH 7 for minutes)
    Further denature proteins
    • Add lysate either a known concentration of proteins or
    cell number equivalent to SDS loading buffer
    • SDS Loading Buffer
    – Buffer (Tris-Cl pH 6.0)
    – 2% SDS
    – 0.1% bromophenol blue
    – 10% glycerol (allows sol’n to sink to bottom of gel wells)
     -mercaptoethanol ( reducing agent)
    • SDS loading gel mixed with lysate is boiled to further
    denature proteins.
    • 1:10 ratio loading buffer to lysate
    SDS-Polyacrylamide Gel
    Electrophoresis of proteins
    • This gel ensures that proteins are
    dissociated into their polypeptide subunits
    and minimizes aggregation.
    • Proteins migrate on gel according to their
    molecular weight therefore if proteins of
    know amounts are added to the gel the
    approximate molecular weight of your
    protein of interest can be found.
    SDS-Polyacrylamide Gel
    Electrophoresis of proteins
    • Protein lysate is now loaded onto a SDS-
    polyacrylamide gel electrophoresis apparatus.
    • This apparatus contains a gel that separates
    protein out according to their molecular weight.
    • GEL
    – 15% 12-43kDa
    – 10% 16-68kDa
    – 7.5% 36-94kDa
    – 5.0% 57-212kDa
    Ingredients in Gel
    • Sodium dodecyl sulfate (SDS)
    • Tris buffer (either glycine or tricine)
    • Acrylamide and NN-bis-acrylamide
    – Forms gel matrix
    • TEMED
    – Catalyst for polymerization (produces free radials from APS)
    • Ammonium persulfate (APS)
    – Source of free radials for polymerization

    Could purchase pre-cast gels if you have the money.


    Transfer of proteins to the membrane
    Western blot-semi-dry transfer of proteins
    After transfer
    • Membrane is washed in a Tris, borate, saline
    solution.
    – Options
    • Added detergent Tween 20 to reduced background in western
    blotting.
    • Block membrane in non-fat dried milk solution.
    Usually 5% w/v. Prevents binding of antibodies to
    membrane.
    – Don’t buy milk from Fisher
    – Safeway brand or Carnation brand are the best
    – Alternatively, use bovine serum albium (BSA) but its
    more expensive (not available in Supermarkets)
    Antibodies
    • Proteins that bind to specific epitopes on specific
    proteins.
    • Two main types used in western blotting
    – Monoclonal
    – Polyclonal
    • Monoclonal are generally produced from mice
    and polyclonal are produced from rabbit or goat
    but other animals also produced polyclonals.
    What antibodies are best for
    western blotting?
    • Both types work for western blotting but
    generally polyclonals are better
    – Monoclonals are to one epitope while
    polyclonals are to many epitopes on the
    protein of interest. If epitope changes upon
    denaturing of the protein it will not recognize
    the protein
    – Each antibody has its own characteristics so it
    is hard to predict what antibodies work well in
    a western and which are bad.
    Primary Antibodies
    • After blocking membrane, add antibodies in
    concentrations recommended by manufacture or
    in dilution series if unknown to blocking solution
    (ie 5% milk).
    • Most antibodies are added as a dilution such as
    1:1000.
    • Incubate overnight at 4oC or 2 hours at room
    temperature (only if it’s a good antibody).
    • Wash in TBS with detergent Tween 20 (TBST)
    Secondary Antibodies
    • Wash at least three times with TBST for 5-10
    minutes each
    • Added antibodies against the animal that the
    primary antibodies were made in.
    • These antibodies are also conjugated with
    enzymes such as horse radish peroxidase
    • The antibodies are added to blocking buffer and
    incubated at room temperature for one hour.
    Detection of your protein
    • After secondary antibody incubation wash
    with TBST as before.
    • Add enhance chemiluminance reagents
    (commercially available) that have
    substrates for HRP that gives a product
    that gives off light energy.
    • Place on autorad film and bands appear.
    Detection
    Primary antibody followed by:
    Radioactive-labelled
    125
    I staphlococcal protein A or streptococcal protein G
    Enzyme-linked secondary antibodies
    -horseradish peroxidase (HRP)
    -alkaline phosphatase-BCIP/NBT
    BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt) and
    NBT (Nitro-Blue Tetrazolium Chloride).

    Chemiluminescent detection-
    HRP catalyzes the oxidation of luminol in hydrogen peroxide.
    Luminol decays by light emission.

    AP catalyzes the dephosphyorylation of adamantyl-1-2-dioxetane


    phosphate, resulting in emission of light.
    Southwestern blot
    Figure 7 Distribution of the 52 kDa protein in various mouse tissues as
    analysed by Southwestern blot analysis

    Biochemical Journal (1998) 329, 623-629 -

    www.biochemj.org

    You might also like

    pFad - Phonifier reborn

    Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

    Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


    Alternative Proxies:

    Alternative Proxy

    pFad Proxy

    pFad v3 Proxy

    pFad v4 Proxy