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  • Protein Separation using various techniques

    Do you have a question regarding separation of proteins using different techniques? Loooking out for help here? No worries, our ISC experts shall respond to yuor question on this Ask Expert page.

    If Protein A=50KDa & pI =3.5
    Protein B=50KDa & pI=7.5
    Protein C=25KDa & pI=5.5
    Protein D=40KDa & pI=5.5
    How will be the banding pattern of these proteins
    1.Considering mass how it separates on SDS PAGE?
    2.Considering only mass how it separates on NATIVE PAGE?
    3.Considering both mass & charge how it separates NATIVE PAGE (Acidic & Basic gel)?
    4.Considering pI how these separates on Isoelectric focusing ?

    Finally which techniques better suites to separate all the above proteins without any of the proteins?
  • Answers

    1 Answers found.
  • There is no single method or technique available for separating and identifying the different proteins. Fortunately, as different proteins have variations in terms of their size, mass, electric charge, solubility etc so using different methods give us confidence in isolating and distinguishing between them. There are many techniques but the most common are centrifugal, chromatography, and electrophoresis ones. These techniques finally help in characterising the protein in terms of the sequence of amino acids in them and finding their 3D structures.

    SDS-PAGE is an electrophoretic method which separates the proteins based on their different masses and is generally used for separating proteins in the molecular mass range 5 to 250 kDa. This method works by making the proteins uniformly charged with negative charges through ionic detergent and the gel starts migrating and separating towards the positive electrode.

    If the molecular weight of the proteins are very near then it becomes difficult to separate them and researchers use different quality gels and their pH value to attain the differentiation to get the band separation on the protein band recording mechanism but sometimes contrast are not obtained and it becomes difficult to separate the nearby proteins. There should be a difference of more than 1 kDa to get some differentiation in the protein segregating through the native or SDS gel.

    If you are interested to know more about these processes in details I would suggest you to visit the bioscience pages where these things are explained in exhaustive ways.

    Knowledge is power.

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