detecting proteins Flashcards
(25 cards)
What is a Chromophore?
The part of a molecule that absorbs light
Chromophores are crucial in determining the color and light absorption properties of molecules.
What does SDS-PAGE stand for?
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SDS-PAGE is a method used to separate proteins based on their molecular weight.
Why do we need to detect biomolecules?
To understand biological processes and monitor cellular activities
Detection of biomolecules is essential in research, diagnostics, and therapeutic applications.
What happens when a light wave strikes an object?
It can be absorbed, reflected, or transmitted
The interaction depends on the properties of the object and the wavelength of the light.
What is the Beer–Lambert law equation?
A = e.c.l
Where A is absorbance, e is the extinction coefficient, c is concentration, and l is path length.
What is absorbance?
The measure of the amount of light absorbed by a sample
It is calculated as log10(transmitted light/incident light).
What types of organic compounds tend to absorb light?
Compounds with ring structures or double bonds
These compounds typically have p orbitals that facilitate light absorption.
Which amino acids act as chromophores in proteins?
- Tyrosine
- Tryptophan
- Cysteine
- Phenylalanine
Each of these amino acids absorbs UV light at specific wavelengths.
What is the extinction coefficient (ε)?
A measure of how strongly a substance absorbs light at a given wavelength
It is used in calculations of protein concentration based on absorbance.
What does the peptide bond absorb strongly at?
214 nm
The absorption varies depending on the environment of the peptide bond.
What is the role of SDS in SDS-PAGE?
It denatures proteins and coats them with negative charges
This allows for separation based on size during electrophoresis.
What is the isoelectric point (pI)?
The pH at which a protein has no net charge
At this point, the protein does not migrate in an electric field.
What is isoelectric focusing (IEF)?
An electrophoretic technique for separating proteins based on their isoelectric point
IEF allows for high-resolution separation of proteins.
What is 2D electrophoresis?
A technique that separates proteins based on isoelectric point and molecular weight
It can analyze hundreds to thousands of polypeptides in a single run.
What is the purpose of staining in gel electrophoresis?
To visualize proteins after separation
Common stains include Coomassie Brilliant Blue and silver stain.
What is Western Blotting?
A technique used to detect specific proteins in a sample
It involves transferring proteins from a gel to a membrane and using antibodies for detection.
What is an epitope?
The molecular structure on an antigen to which an antibody binds
Epitopes are typically made up of several adjacent amino acids.
What are primary antibodies?
Antibodies raised against a specific target antigen
They are usually produced in animals like mice or rabbits.
What is a secondary antibody?
An antibody raised against the host species of the primary antibody
It is used to amplify the signal in detection methods.
What is the role of a loading control in Western Blotting?
To ensure that the change in expression of the target protein is real
Common loading controls include Actin, GAPDH, and Tubulin.
What is the detection limit of Coomassie Brilliant Blue?
10 to 100 ng
This indicates the sensitivity of the staining method for protein detection.
What is the detection limit of silver staining?
Less than 1 ng of protein
Silver staining is highly sensitive compared to other staining methods.
What is the purpose of using antibodies in biomolecule detection?
To achieve specific detection of biomolecules
This allows for the identification of a single type of biomolecule among a mixture.
What is the function of ethidium bromide in DNA visualisation?
To bind to DNA and allow for its detection under UV light
The detection limit is between 0.5 to 5.0 ng/band.
