4.4 LEC Flashcards
What detection system uses 32P-labeled probes to detect hybridization through autoradiography?
Radioactive detection
What must be done after hybridization to ensure that only completely hybridized probes remain on the blot?
Wash under stringent conditions
What are two common nonradioactive labels used in detection systems?
Digoxigenin and Biotin
Which enzyme is typically conjugated to anti-digoxigenin or streptavidin for nonradioactive detection?
Alkaline phosphatase (AP)
What detection system involves the release of light by phosphorylation of dioxetane?
Chemiluminescent detection
Which detection method allows direct observation of color developing on the membrane?
Chromogenic detection
What is a key factor in achieving a successful blotting method?
High signal-to-noise ratio
What agents are used to prevent nonspecific binding in nonradioactive detection systems?
Blocking agents
What type of enzyme conjugates, in addition to alkaline phosphatase, may also be used for signal generation?
Horseradish peroxidase (HRP)
What are two common substrates used in nonradioactive detection that produce chemiluminescent or color signals?
Dioxetane and Tetrazolium dye derivatives
What can a Southern blot detect by comparing restriction patterns of samples?
Gene locus restriction patterns
What type of control is included in Southern blot analysis to ensure accuracy despite low expression levels?
Internal standardized control
What issue can arise in Southern blot assays when a probe binds to multiple similar sequences?
Cross-hybridization
What can Southern blot analysis not detect unless it affects a restriction enzyme site?
Tiny deletions, insertions, or single-nucleotide differences
Which blotting technique is used for analyzing gene expression, transcript size, and protein modifications?
Northern or Western blot
