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Western Blot Dictionary

Anionic

A negatively charged ion. Attracted to the anode in electrolysis.

Cathode

A cathode is a negatively charged electrode. It can act as source of electrons or electron donor.

 

APS

Ammonium Persulphate.  APS initiates the polymerization of the acrylamide mix. It provides free radicals necessary for polymerization to take place.

 

BCA

Bicinchoninic acid assay. Biochemical spectroscopic assay for determining the total concentration of protein in solution.

Blocking

The western blot membrane is blocked to prevent non-specific binding.

Bradford Assay

A spectroscopic protein assay to measure the concentration of protein in a solution. Assay is dependent on the amino acid composition of the measured protein.

Chemiluminescence

Chemiluminescence is the production of light from a chemical reaction. Two chemicals react to form an excited intermediate, which breaks down releasing some of its energy as photons of light to reach its ground state.

Denature

The process by which a protein loses it quaternary, tertiary and secondary structure.

Detergent

A detergent has unique properties which disrupt hydrophobic-hydrophilic interactions in biological samples.

 

Disulphide bonds

A disulphide bond is a covalent bond derived from two thiol groups. Disulphide bonds can help in protein folding following oxidation.

ECL

Enhanced chemiluminescence. To be used when antibodies are labelled with HRP, a chemiluminescent substrate is added such as luminol and oxidising agent such as H2O2 which form excited intermediates, emission at 540 nm.

Epitope

Part of an antigen recognised by the immune system.

Extraction buffer

Also referred to as lysis buffer used to extract protein from cells.

Fluorophore

A fluorescent chemical compound which can emit light upon excitation.

Gel Electrophoresis

Gel electrophoresis used to separate mixtures of DNA, RNA, or proteins according to molecular size.

Ionic

In ionic bonding, atoms transfer electrons to each other. Ionic bonds require at least one electron donor and one electron acceptor.

Lameli Buffer

Lameli Buffer contains beta-2-mercaptoethanol or dithiothreitol (DTT) which act to reduce disulphide bonds before they adopt the random-coil configuration and in turn denatures the protein. Lameli buffer also has a SDS component which provides the negative charge necessary for gel electrophoresis, in addition to glycerol to make the sample denser.

Loading buffer

e.g Lameli Buffer is added to your protein to enable visualisation and migration through the gel.

Lysis Buffer

Used to extract protein from cells.

Nitrocellulose Membrane

A membrane used for western blot transfer, has a high protein-binding affinity and is compatibility with multiple methods of detection. Proteins are immobilized via hydrophobic interactions.

Non-specific binding

Non-specific binding refers to the binding of a ligand to something other than its designated receptor.

Native state protein

The native state of a protein refers to a protein in its properly folded state with operative functionality.

Primary antibody

A primary antibody is an immunoglobulin that specifically binds to a protein of interest for purifying or detecting and measuring it.

Probe

Used to identify and quantify the binding of the secondary antibody to the primary antibody and determine the amount of protein in your sample.

PVDF

Polyvinylidene difluoride (PVDF) is a highly non-reactive thermoplastic fluoropolymer produced by the polymerization of vinylidene difluoride. non-specific affinity for amino acids.

Qualitative Assay

Qualitative assays help to identify the presence or absence of the analyte being tested.

Quantitative Assay

A quantitative assay measures the presence, amount, or functional activity of the analyte.

Resolving gel

The resolving gel is the bottom and larger portion of the gel. It is poured first.

RIPA Buffer

RIPA (Radio Immuno Precipitation Assay) buffer is used to lyse and extract protein from cultured cells.

SDS

Sodium dodecal sulfate (SDS) is an anionic detergent that partially denatures and coats your protein.

SDS-PAGE Gel electrophoresis

SDS-PAGE Gel electrophoresis uses a polyacrylamide gel as a support medium and SDS to denature the proteins.

Secondary antibody

Secondary antibodies bind to the primary antibody. Secondary antibody binding enables the detection and purification of target antigens. The secondary antibody must have specificity for the antibody species and isotype of the primary antibody being used and generally is conjugated to enable chemiluminescent or fluorescent detection.

Semi-Quantitative

Yielding an approximation of the quantity or amount of a substance; between a qualitative and a quantitative result.

Species specific

Refers to the action of an antibody or drug being limited in action or effect to a particular species.

Stacking gel

The stacking gel is poured on top of the resolving gel – the combs are placed into this gel. The stacking gel is needed to concentrate/pack all the proteins in one band, so that they will start migrating in the running gel all at the same time.

TEMED

TEMED acts as a catalyst to speed up the polymerization reaction of the gel. Once you add the TEMED poor the gel immediately.

 

Transfer

Refers to the transfer of your separated protein to membrane which will probed and the amount of protein in your sample determined.

Tris

Tris is the buffer of choice to regulate gel’s pH. Stacking buffer is usually pH 6.8 and resolving gel is pH 8.8.