L13: Electrophoresis Flashcards
What is electrophoresis?
Migration of ions in an electric field, widely used for separation of biological macromolecules (proteins)
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What is the electric force of an ion (q) expressed by?
F(electric) = qE
What is the electrophoretic migration of an ion through the solution expressed by?
F(friction) = vf
Does the forces on the ion balance in a constant electric field?
Yes
qE = vf
How is electrophoretic mobility (μ) expressed?
μ = v/E = q/f
Most common gels that are used and what they are used for?
Polyacrylamide for proteins & agarose for nucleic acids
Mesh size uses
Large -> larger pores -> better resolution for larger molecules
Smaller -> smaller pores -> better resolution for smaller molecules
What is gel electrophoresis used for?
Separating macromolecules based on size & charge
What does SDS-PAGE stand for?
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
How does gel electrophoresis separate biological macromolecules?
Based on their molecular masses
What is the function of beta-mercaptoethanol in SDS-PAGE?
To break disulfide bonds in proteins
What is SDS?
Negatively charged detergent that binds to proteins, unfolds them & gives them a uniform charge-to-mass ratio
What is the purpose of using Coomassie Brilliant Blue in protein visualization?
To stain proteins for visualization
How do proteins migrate during SDS-PAGE electrophoresis?
Based on their molecular masses
What role does sodium dodecyl sulfate (SDS) play in SDS-PAGE?
To denature proteins and add negative charge
What factors influence protein electrophoresis?
Molecular mass, protein charge, and isoelectric point
What methods can be used to visualize proteins after electrophoresis?
Silver Staining
What is agarose gel electrophoresis used for?
DNA & RNA separation
Gel staining of agarose gel electrophoresis
Ethidium bromide (fluorescent under UV light)
What is the significance of the isoelectric point (pI) in protein separation?
It is the pH at which a protein has NO net charge and can be separated from other proteins
WHat is separation based on in isoelectric focussing (IEF)?
Separated based on their isoelectric point (pl)
How is the pH gradient in isoelectric focussing formed?
Mixture of polyampholytes (small multicharged polymers)
What is the importance of mesh size in polyacrylamide gel electrophoresis?
To determine the size of molecules that can pass through the gel
How does two-dimensional gel electrophoresis enhance protein separation?
By separating proteins based on charge, then size
1) isoelectric focussing
2) SDS-PAGE
Advantage of 2D gel electrophoresis
Resolves proteins with identical molecular weight but different charges
4 ways to visualise proteins
1) Coomassie brilliant blue (CBB)
2) Silver staining
3) Ponceau red
4) Immunoblotting (western blotting)
What is the process of blocking in immunoblotting, and why is it important?
Saturating the membrane to prevent non-specific antibody binding
Use of ponceau red
Validating transfer to nitrocellulose after blotting
What does immunoblotting need?
Needs primary antibody (binds target protein) & secondary antibody
What is the purpose of immunoblotting in protein detection?
To detect specific proteins
Function of primary/secondary antibody binding in immunoblotting
Primary: recognises target protein
Secondary: recognises primary antibody
Steps in the detection of proteins by immunoblotting
1) Blot proteins from the gel onto nitrocellulose
2) Block unoccupied binding sites with casein
3) Incubate rabbit antibody to protein of interest (primary antibody)
4) Wash & incubate with enzyme-linked goat anti-rabbit antibody (secondary antibody)
5) Assay linked enzyme with colorimetric reaction
What is the function of chemiluminescence substrate in the detection process?
To produce light for detection
What is the significance of using nitrocellulose in immunoblotting?
To act as a solid support for proteins
What role does the primary antibody play in the immunoblotting process?
To specifically bind to the target protein
How does the secondary antibody contribute to the detection of proteins?
It provides an enzymatic tag for detection
What type of enzyme is commonly linked to the secondary antibody in immunoblotting?
An enzyme
What is the importance of blocking unoccupied binding sites on nitrocellulose?
To reduce non-specific antibody binding
Why might multiple chromatographic columns be needed for protein purification?
To separate proteins based on different properties for higher purity
What factors determine the choice of purification method for a specific protein?
The protein’s properties and the contaminants present
What is the relationship between molecular weight markers and protein separation?
Markers estimate the sizes of separated proteins
What is the purpose of affinity chromatography in protein purification?
To isolate a protein based on binding affinity
How can the specific activity of a protein be calculated during purification?
Total activity / Total protein
