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Preservation of protein structure in electroblotting

  1. May 21, 2014 #1

    I am reading about the Western blot technique, and I am looking for clarification of the transfer step described in the wikipedia article and in this video walkthrough. It is not clear to me why the proteins should keep their shape needed for the probing step rather than breaking or deforming. Is this because the strength of bonds within the proteins are stronger than the electrical force pulling them to the nitrocellulose membrane? Or is the whole protein pulled uniformly? My knowledge of physics involved in protein structure is virtually null.
  2. jcsd
  3. May 21, 2014 #2


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    The protein should be denatured during the transfer step. The SDS-PAGE step beforehand should denature the protein (b/c the presence of SDS and boiling the sample in the presence of reducing agent).
  4. May 24, 2014 #3
    Rethinking it, I guess I might have asked why the structure is preserved in the electrophoresis...
    So denaturing preserves the primary structure. The probing is done on linear epitopes then?

    I think my real question was what kinds of forces act on and within proteins and polymers generally such that they primarily(?) experience conformal changes by the action of enzymes and not by other forces. Maybe I should pick up a book on physical chemistry...

    Thanks for the response :)
  5. May 24, 2014 #4
    Proteins aren't rigid structures and protein dynamics are now thought to play a large part in the function of enzymes and such. You can try to google terms like 'induced fit' to see that enzymes do indeed wobble around and change conformation quite a bit to perform catalysis.

    Without writing a book chapter I will recommend that you check out any biochemistry textbook to get an idea about protein structure organization. Physical chemistry texts won't give you much insight into proteins specifically but I'd imagine a book written more for physical biochemistry may have some interesting takes on the thermodynamics and kinetics of protein structure. I guess it depends on what level if math and physical science you are comfortable with. Biochemistry can range from physical chemistry applied to biological molecules to a more descriptive biological approach with mostly text, diagrams and very little math/physics.
  6. May 24, 2014 #5


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    Yes, during SDS-PAGE, the protein is denatured but the primary structure (i.e. the sequence of amino acids making up the protein) is preserved. Whereas the secondary, tertiary, and quaternary structure depends mostly on non-covalent bonds and interactions (e.g. hydrogen bonds, electrostatic interactions, van der Waals contacts, hydrophobic effect), the primary structure of the protein is maintained by covalent peptide bonds that are not broken up during the SDS-PAGE sample prep (the exception here is that the formation of covalent disulfide bonds can contribute to the tertiary and quaternary structure of proteins, but these covalent bonds are broken by reducing agents during the sample preparation for SDS-PAGE).

    Because the protein are denatured, most of the antibodies that work well for Western Blotting target linear epitopes (often these antibodies are raised by injecting animals with short peptides). For this reason, many epitopes that work well for Western blotting (where the proteins are denatured) may not work well in applications where the protein is intact (e.g. ELISA, immunofluorescence) and vice versa.
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