Molecular and Immunological Investigation Techniques PDF

**[Molecular and immunological investigation techniques - 2.4]** Nucleic acids: - - - - Protein - - - - - - **[Immunostaining]** Uses AB to stain cells Direct = uses a primary AB with fluorophores, specific but harder to detect Indirect = less specific (some background)...

**[Molecular and immunological investigation techniques - 2.4]** Nucleic acids: - - - - Protein - - - - - - **[Immunostaining]** Uses AB to stain cells Direct = uses a primary AB with fluorophores, specific but harder to detect Indirect = less specific (some background), easier to detect Immunocytochemistry - Immunohistochemistry Immunofluorescence Antibodies 1.  2. More specific, less variation 3. Generated in lab  Flow cytometry Light scatters as the green laser interrogates the cell Direction of light scattered correlates to cell size and granularity Can measure: - - - - - - Limitations: - - -   Dot plot of FS vs SS Each dot represents a single cell The characteristic position of different cell populations is determined by differences in cell size and granularity  Fluorescence activated cell sorting (FACs) Specialised type of flow cytometer that sort a heterogenous mixture of cells based on specific light scattering and fluorescent characteristic of cell ELISA - - - -  Direct ELISA - - Indirect ELISA - - Sandwich ELISA - - Competitive ELISA - - Multiplex - - In situ hybridisation - - - -  PCR - - -  Reverse transcriptase - quantitative polymerase chain reaction (RT-qPRC) - - 1. 2. 3. PCR limitations: - - - - Electrophoresis - - Agarose gel = used for DNA Polyacrylamide gel = used for proteins Large travel to top Small travel to bottom Post gel analysis DNA - ethidium bromide Protein - coomassie blue, silver nitrate, spyro ruby Blotting techniques - Western blotting = quantitative protein analysis - - Southern blotting = labelled nuclei probes that recognise complementary DNA sequences Northern blotting = labelled nuclei probes that recognise complementary RNA sequences Detection systems based on luminescence or radioactivity can be used to detect protein or nucleic acid targets, revealing original migration position and quantity of target  Mass spectrometry - - - - Methods: - - - - 1. 2. 3. 4. - Limitations: - - - Microscopy - -  - Eg Fluorescence in situ hybridisation - - **Summary** RT PCR = determines genetic transcription of a particular protein Western blot = to determine if gene transcription leads to the transcription of a functioning protein ELISA - to measure the amount of the transcribes protein secreted out of the cell