How do superparamagnetic micro-beads attract proteins in immunoprecipitation?

  • Thread starter Bararontok
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Immunoprecipitation is a technique used to separate proteins from organic samples for investigation. Initially, rough and porous sepharose and agarose polymers were utilized to isolate proteins and they used adhesion to their rough surfaces, gaps and hollow spaces inside them to capture the proteins but these smooth superparamagnetic beads use their magnetic fields for protein capture. How is it possible to use magnetism to attract proteins? Are proteins magnetic like metal?
 

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  • #2
DrDu
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The proteins do not bind to the beads because of the beads magnetism. Rather the magnetism of the beads is used to separate them from the solution via applying a magnetic field once the precipitation is completed. With non-magnetic beads you would have to use e.g. centrifugation.
 
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If magnetism cannot attract the proteins, then how do the proteins bind to the magnetic beads if the magnetic beads are much smoother than the sepharose and agarose polymers?
 
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Ygggdrasil
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Sepharose, agarose, and paramagnetic beads are not supposed to bind protein on their own. Rather, these beads are first chemically crosslinked to antibodies that have been made to bind only the protein you are interested in immunoprecipitating (hence the name immunoprecipitation as the technique relies on antibodies [i.e. immunoglobulins]).
 
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Then how do the antibodies link to the proteins and how do the sepharose, agarose and paramagnetic beads bind to the antibodies? What specific physical mechanism causes the binding? Is it caused by the mechanical interlocking of molecules or by the binding of molecules via the exchange of electrons similar to various chemical bonds?
 
  • #6
Then how do the antibodies link to the proteins
The reason for the existence of antibodies is to bind to their antigens.
en.wikipedia.org/wiki/Antibody

and how do the sepharose, agarose and paramagnetic beads bind to the antibodies? What specific physical mechanism causes the binding? Is it caused by the mechanical interlocking of molecules or by the binding of molecules via the exchange of electrons similar to various chemical bonds?
Agarose/sepharose is attached to antibodies via covalent interactions.
 
  • #7
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The reason for the existence of antibodies is to bind to their antigens.
en.wikipedia.org/wiki/Antibody
The wikipedia article states that each antibody has a unique paratope that matches the epitope of a specific antigen so this means that these uniquely structured paratopes and epitopes are used for the mechanical interlocking of molecules on the antibodies and proteins to bind them together like some type of mechanical coupling.

Agarose/sepharose is attached to antibodies via covalent interactions.
Then what type of interaction binds the paramagnetic beads to the antibodies?
 
  • #8
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The interaction between antibodies and antigens can depend on more than just steric factors (the "mechanical" picture you seem to have in mind) - one also has to consider electrostatics, hydrophobic interactions, and other chemical/physical interactions.

The magnetic cores of these microbeads (typically iron oxide particles) are initially coated with a polymer shell as I understand the manufacturing process (so it just wraps around the iron oxide particle, and doesn't interact with the iron oxide), and then the outer surface of the polymer shell is chemically modified to make, for example, covalent bonds with the antibody or an intermediate coating that is more biologically inert like dextran or agarose. There are a number of chemical modification schemes that are out there that can link together two components - one popular one is the EDC/NHS coupling scheme, which has been applied in a variety of contexts.
 
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Ygggdrasil
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The wikipedia article states that each antibody has a unique paratope that matches the epitope of a specific antigen so this means that these uniquely structured paratopes and epitopes are used for the mechanical interlocking of molecules on the antibodies and proteins to bind them together like some type of mechanical coupling.
To add to Mike's post, please see the following wikipedia articles for more information about the types of forces involved in protein-protein interactions:

https://en.wikipedia.org/wiki/Intermolecular_force
https://en.wikipedia.org/wiki/Hydrophobic_effect
 
  • #10
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So in conclusion, the antibodies and proteins bond together through intermolecular and hydrophobic interactions and the super-paramagnetic bead is coated with polymers, dextran or agarose to enable the bead to form a covalent bond with the antibody.
 

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