Protein Detection Techniques – Western Blotting, ELISA & Multiplex Assays

A comprehensive set of vocabulary flashcards covering key terms, components and principles of Western blotting, ELISA and multiplex immunoassay techniques.

Polyacrylamide Gel Electrophoresis (PAGE)

Technique that separates proteins in a polyacrylamide matrix under an electric field.

SDS-PAGE

Denaturing PAGE where sodium dodecyl sulphate coats proteins with negative charge and linearises them, separating mainly by molecular weight.

Native PAGE

Non-denaturing PAGE that separates proteins in their native state based on charge density, size and shape, preserving activity.

Reducing Agent (e.g., 2-mercaptoethanol)

Chemical added to break disulfide bonds and fully denature proteins before SDS-PAGE.

Western Blotting (Immunoblotting)

Method that combines PAGE separation, electroblot transfer to a membrane, and antibody-based detection of specific proteins.

Electroblotting

Electric-current driven transfer of proteins from a gel onto a nitrocellulose or PVDF membrane.

Nitrocellulose Membrane

Common porous membrane used in Western blots to bind transferred proteins.

Polyvinylidene Fluoride (PVDF)

Hydrophobic membrane alternative to nitrocellulose with high protein-binding capacity for Western blots.

Blocking (Western Blot)

Incubation of membrane with irrelevant protein (e.g., 5 % milk, BSA) to cover unoccupied sites and reduce non-specific antibody binding.

Primary Antibody

Antibody that specifically binds the target protein on a blot or ELISA plate.

Secondary Antibody

Enzyme- or tag-conjugated antibody that recognizes the primary antibody and produces measurable signal.

Chemiluminescent Detection

Signal system where enzyme reaction emits light, used frequently in Western blot visualization.

Housekeeping Protein

Endogenous protein (e.g., GAPDH, β-actin) used to normalize Western blot signals for semi-quantification.

Densitometry

Measurement of band intensity on a Western blot for quantitative analysis.

Radioimmunoassay (RIA)

Highly sensitive assay where radiolabelled antigen competes with sample antigen for antibody binding.

Enzyme-Linked Immunosorbent Assay (ELISA)

Plate-based immunoassay using enzyme-labelled antibodies to detect and quantify analytes.

Direct ELISA

Format where an enzyme-labelled antibody binds directly to the immobilised antigen.

Indirect ELISA

Format using an unlabeled primary antibody and an enzyme-labelled secondary antibody for detection.

Sandwich ELISA

Assay where antigen is ‘sandwiched’ between a capture antibody on the plate and a detection antibody.

Competitive ELISA

Format where sample antigen competes with labelled antigen for limited antibody binding sites.

Plate Coating (ELISA)

Immobilisation of antigen or capture antibody onto microplate wells.

Surface Blocking (ELISA)

Addition of irrelevant proteins to occupy unbound sites and prevent non-specific binding.

Standard Curve

Serial dilution of known antigen used to convert ELISA or multiplex signals into precise concentrations.

Quantitative ELISA

ELISA interpretation that provides exact antigen concentration via a standard curve.

Qualitative ELISA

Assay interpretation giving a yes/no answer on antigen presence.

Semi-Quantitative ELISA

Comparison of relative antigen levels across samples based on signal intensity.

Colorimetric Detection

ELISA read-out producing a colored product (e.g., TMB) measured by absorbance.

Chemifluorescent Detection

ELISA read-out where excited molecules emit photons, detected as fluorescence.

TMB (3,3',5,5'-Tetramethylbenzidine)

Common chromogenic substrate for HRP in colorimetric ELISAs; turns blue then yellow on acid stop.

Plate Reader

Instrument that measures absorbance, fluorescence or luminescence in microplate assays like ELISA.

Multiplex Immunoassay

Technique that simultaneously measures multiple analytes in one sample, e.g., Luminex assays.

Luminex Technology

Bead-based multiplex platform using internally dyed beads and flow cytometry to identify and quantify analytes.

Bead Region

Unique fluorescent signature of a Luminex bead designating a specific analyte.

Capture Antibody (Multiplex)

Antibody coupled to a bead that binds its specific target analyte from the sample.

Biotinylated Detection Antibody

Secondary antibody labelled with biotin, enabling PE-streptavidin binding for fluorescence read-out.

Phycoerythrin (PE)

Fluorescent dye conjugated to streptavidin for detecting biotinylated antibodies in Luminex assays.

Dual-Laser Flow Cytometer

Instrument that simultaneously identifies bead colour (analyte type) and PE intensity (analyte amount) in Luminex assays.

Multiplex Advantage

Ability to quantify many targets concurrently, saving sample volume, time and cost versus single ELISAs.

Multiplex Limitation

Requires specialised equipment and careful antibody pair validation to avoid cross-reactivity.

Chemiluminescence (Transient)

Short-lived light emission used in highly sensitive Western blot or ELISA detections.

Electrophoresis Tank (Gel Box)

Apparatus holding buffer and electrodes to run PAGE separations.

Wet Tank Transfer

Protein transfer technique where gel and membrane are submerged in buffer between electrodes.

Semi-Dry Transfer

Protein transfer method using minimal buffer between stacked filter papers and membranes.