L5 Non-competitive immunoassays Flashcards
Critically discuss the basis of immunoblotting. Critically discuss the use of immunocytochemical/histochemical techniques. Explain and understand how immunoassays may be applied in molecular bioscience and biomedical research.
What are the two immunoblotting techniques?
Dot blot.
Western blot.
What are the four immunoassay microscopy techniques
Immunohistochemistry.
Immuno-cytochemistry/-fluorescence
Proximity ligation assays
Immunogold electron microscopy
What do both dot blotting and western blotting techniques involve?
What is the difference?
Involve immobilisation of sample onto membrane filter (usually nitrocellulose or PVDF)
Probing of the membrane with antibodies for detection.
Difference is dot blot only detects presence/absence of antigen, and western blot indicates molecular weight also.
Can dot blotting be used quantitatively?
Yes, using densitometric analysis of spot intensity using purified antigen for calibration.
Protein arrays evolved from which immunoblot technique?
Evolved from traditional dot blot, but much higher throughput.
What two basic techniques are involved in western blot?
Most commonly SDS-PAGE followed by immunodetection.
What is the role of SDS in PAGE and what does this mean for some antigens and their antibody affinity?
SDS is a negatively charged detergent that binds all over a protein when boiled alongside a reducing agent (e.g. beta-mercaptoethanol) Gives protein a net negative charge.
This prevents charge of native protein does not influence movement over SDS-PAGE gel and ensures molecular weight is the only distinguishing factor.
Means that discontiniuous antigen epitopes (where antibody recognises tertiary structural epitope) may not be recognised by antibody. Continuous epitopes must be used.
What are coomassie blue and silver staining used for in SDS-PAGE?
Both used to visualise protein in the gel. Silver much more sensitive.
Why may denaturing conditions be omitted during PAGE?
Due to denaturing disulphide bonds being broken, affects shape of protein sample, if this is critical.
e.g. antibodies are affected by denaturing due to disulhpide bonds and behave differently on PAGE.
How can charge be incorporated to PAGE if neccessary?
Taking advantage of isoelectric point of amino acids, proteins have isoelectric point and a pH that they exist at neutral charge.
Doing a 2D-PAGE where sample is first ran through gel tube that separates based on pH, then perpendicularly ran over regular SDS-PAGE.
Gel is generated that from left to right separates proteins based on charge, then from top to bottom separates proteins based on size.
What is the basic definition of western blotting?
An immunoblotting technique often used to detect the presence of a target protein in a sample.
What are the steps of western blot?
What does it allow?
SDS-PAGE (or 2D-PAGE) first used to separate sample of interest then gel is electrophoretically transferred to a membrane (nitrocellulose or PVDF).
Once samples are immobilised on membrane, an antibody can be used to detect a protein of interst in the sample mixture.
It allows determination of antigen presence, molecular weight and/or charge (thereby post-translational modifications). If combined with other techniques (cell fractionation) localisation too.
What is the purpose of the botting (transfer) apparatus?
Transfer proteins from the gel to a positively charged nitrocellulose membrane (or pvdf) using electrophoresis.
What are the two forms of blotting during the transfer between PAGE and detection?
Semi-dry blotting where transfer buffer is applied to filter paper sandwich.
Wet blotting where whole assembl submerged in transfer buffer.
Briefly describe the blotting (transfer) apparatus
Four layers. Top and bottom layers blotting paper.
Second from top layer is SDS-PAGE gel. Beneath that is the transfer membrane.
Top is negatively charged and bottom membrane is negatively charged.
What is the transfer buffer?
Tris/glycine based and can contain SDS or methanol. Usually pH 8.3 but varies depending on pI (isoelectric charge) of protein of interest.
How does detection occur at the final step of western blotting?
Proteins have been bound to a membrane and can be ‘probed’ with antibody to detect protein of interest.
Block of non-protein bound sites with incubation with BSA or milk powder.
Then incubate with primary antibody specific to antigen of interest.
Washed to remove unbound primary antibody.
Reveal reactivity (depends on conjugate)
What are the three general ways that detection can be conveyed by antibodies in immunoblot?
Enzymes such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).
Chromogenic substrates such as AP with coloured substrates, HRP with DAB and peroxide, or dark coloured insoluble reaction products.
Radiolabelling by radiolabelled antibody or protein A/G, or detection by autoradiography.
How can enhanced chemiluminescence (ECL) be used for detection in immunoblot
HRP labelled secondary antibody, with HRP activity coupled to oxidation of luminol. Light released during this reaction detected by X-ray film or using a CCD camera based imaging system.
Four controls can be used during immunoblotting. Two positive and two negative. What are they?
Positive control lysate, using enriched source of antigen to confirm antibody works.
Negative control lysate with knockout or depleted source of antigen.
Using no primary antibody or control Ig in place of primary antibody negative control, controls for non-specific activity of secondary antibody.
A positive control can be using another antibody/antigen of known reactivity.
The two issues associated with western blotting are poor transfer and poor Ab detection. How can they be overcome?
A lot of troubleshooting may be required for Western blot, many steps that need optimising for different antibodies and samples.
Poor transfer can be overcome by decreasing acrylamide concentration, transferring usining wet blotting (increases transfer efficiency of high weight proteins).
Poor detection can be overcome by using different antibody concentrations or incubation temperatues. Using different blocking agents. Using loading control antibody e.g. actin.
What is the main purpose of cell/tissue staining microscopy?
Using labelled antibodies which localise to target. Reveals sub-cellular localisation or tissue location of specific antigens.
What are the three main assays for cell/tissue staining microscopy?
Immunohistochemistry staining. Immunocytochemistry or proximity ligation assays.
What is the general procedure for staining microscopy?
FIX - Sample is fixed (frozen in place).
EXTRACT - Lipid removed using neutral detergent.
BLOCK - BSA/milk etc
Primary antibody inc. then wash.
Conjugated secondary antibody inc. then wash.
REVEAL - Light microscope or fluroescence microscope etc
A main problem with IHC is antigen masking resulting in false negative results. What are possible solutions for this?
Antigen must be exposed/retrieved.
Changing method of fixation.
Protease treatment
Heat treatment
Pre-extraction
Trying different antibody (e.g. poly/mono)
IHC has many uses as a diagnostic and research tool, name a few
Identify biomarkers of disease and subcellular/tissue localisation of POI to optimise treatment regimes and stratify patients.
Immunofluorescence (IF) is a type of immunocytochemistry. What reasons would it be used in research?
Used for protein detection, localisation and co-localisation (studying of multiple proteins of interest).
What advantage does confocal microscopy have over conventional?
Higher resolution, able to control photo depth of field, eliminate background information.
Focal place much thinner.
What is a proximity ligation assay used for?
Used for localised detection and manipulation
What is the fundamental difference between immunogold electron microscopy and other immunocytochemistry techniques?
What advantage does this convey?
The antibodies/probes are labelled with colloidal gold.
This increases electron scatter to give high contrast ‘dark spots’
How does immunogold electron microscopy work?
Same as other IF techniques, but secondary antibody is conjugated with gold nanoparticles.
