Protein Detection Techqniues Flashcards
Western Blotting (Immunobloting): Principles
A laboratory method using antibodies to detect specific protein molecules in a mixture of proteins.
Combines:
▪ Separation of proteins in a semi-solid Polyacrylamide gel (PAG) using PAG electrophoresis (PAGE)
▪Electroblotting of the separated proteins onto a solid membrane (nitrocellulose or polyvinylidene fluoride (PVDF)
▪ Detection of target proteins by specific antibodies
Native Gel Electrophoresis
Proteins separated according to their native charge
SDS-PA
Proteins separated according to their molecular weight:
1)proteins are denatured
2)proteins are native (not denatured )
Native PAGE
Separates the proteins in their native state without pre-treatment with • Heat
• Sodium dodecyl sulphate (SDS) which attaches a negative charge
• Denaturants which break the tertiary and quaternary protein structure
The proteins are separated based on their:
• Charge density (charge to mass ratio) • Size
• Shape
The Native PAGE is carried out at 4C
The proteins retain their function after the Native PAGE
SDS-PAGE: Sample preparation
Reducing agents (e.g. 2-mercaptoethanol) could be used to reduce disulphide bridges and dissolve tertiary and quaternary structures
SDS: attaches to protein molecules, charges them negatively and linearises the protein chains
Western Blotting: Detection
Blocking of the membrane with irrelevant for the assay protein, e.g. 5% skimmed milk powder (contains casein) in PBS or 1% bovine serum albumin in PBS (like in ELISA)
→Exposure of membrane to primary antibody → Washing
→Exposure of membrane to secondary antibody
→ Washing
→Detection (often chemiluminiscence)
Western Blotting: Data Analysis
• Theintensityofthetargetandhousekeeperbandsisdetectedbydensitometry
• The protein expression of the target is normalised to the protein levels of the reference protein (often GAPDH, b-actin, tubulin, etc.)
Radioimmunoassay (RIA)
▪Very sensitive method for quantification of antigens (requires a standard curve)
▪Principle of RIA: Antigen-antibody reaction in which a trace amount of the radiolabeled antigen competes with endogenous antigen for limited binding sites of the specific antibody against this
antigen.
▪Advantages: highly sensitive, fast, can be automated ▪Disadvantage: uses radioactivity for detection
▪Applications: used to detect a variety antigens, antibodies, hormones, drugs, vitamins etc.
Enzyme-Linked Immunosorbent Assay (ELISA)
Described by E. Engvall and P. Perlmann in 1971:
Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971 Sep; 8: 871-4.
ELISA:
▪ Fast and safe method for detection and quantification of a particular molecule in a complex mixture by the use of specific antibodies
▪ A type of labelled immunoassay where tagged (labelled) antibodies are used to quantify target molecules
▪ Can be automated
ELISA substrates and signal detection
- Chemiluminiscent: emit light as a result of a chemical reaction
Most sensitive, but reactions are transient - Chemifluorescent: emission of photons when electrons shift from an “exited” to “ground” energy state
- Colorimetric: formation of a soluble colourful substance, which absorbs light at a particular wavelength in the visible spectrum
Most often used in research
Main steps in ELISA
1.
2.
Coating / Capture:
Main steps in ELISA
3.
4.
direct or indirect mobilisation of antigens to hard surface (microplate well)
Surface blocking:
addition of irrelevant protein(s) to cover all unsaturated surface-binding sites of the microplate well
Probing / Detection:
incubation with antigen specific antibody that binds specifically to the antigen
Signal measurement:
detection of the signal generated via a direct or a secondary tag (label) on specific antibody
Extensive rinsing of the wells with wash buffer after steps 1-3 is essential to prevent false positive results
ELISA formats
I. Direct ELISA:
Direct binding of a labelled detection antibody to target molecule
II.
1. 2.
Indirect ELISA:
Detection antibody binds to target molecule
Detection antibody is recognised by a specific enzyme-labelled secondary antibody
Sandwich/Capture ELISA
Sandwich: Target molecule “sandwiched” between 2 detection antibodies Capture: Recombinant antigen is used to capture target antibody
-competitive Elisa
ELISA data interpretation
The ELISA assay can yield 3 types of data:
Quantitative (predominant use): Use of a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in the samples.
Semi-Quantitative: Comparison of the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.
Qualitative: Provides a “yes” or “no” answer, indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen, or an unrelated control antigen.
Luminex® Multiplex assay
▪ Allows the simultaneous analysis of multiple proteins in single samples from a broad range of biological sources.
▪ Combines the efficiencies of multiplexing with the accuracy, sensitivity, reproducibility, and simplicity of ELISA.
• Efficient: simultaneously analyse multiple proteins using only up to 50 μL of sample
• Economical: help save time and costs compared to Western blot or ELISA • Simple: easy operation with streamlined protocols
Principles of Luminex® Multiplex Technology I
▪ Polystyrene or paramagnetic beads are internally dyed with red and infrared fluorophores of differing intensities.
▪Each dyed bead is given a unique number (bead region), allowing the differentiation of one bead from another.
▪Individual bead sets are coated with a capture antibody against one specific analyte
