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.
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).
