Theory L7 - Biochemical Perspective Flashcards
Why use objective measures?
They can be good for verifying subjective measures
Name some objective methods.
Histology (immunohistochemistry) Enzyme immunoassays (ELISA, EIA, RIA) Protein Analysis (Western Blot) Gene Expression (Northern Blot) DNA Sequence (Southern Blot) PCR RT-PCR
What are the steps in histology?
Fixing - Chemical - to preserve from decay
Processing - dehydrating, clearing out and infiltration -
Embedding - The technique of placing cells or tissue in a supporting medium so that thin sections can be cut.
Sectioning
Staining
- important because the tissue can be easily damaged. Cell membranes are very sensitive to oxygen and can leak/break.
- it is possible to lose what you’re interested in. Artefacts could also be created.
what are examples of histology?
CRESYL VIOLET
LUXOL FAST BLUE
GOLGI STAIN
AMYLOID STAIN
What is cresyl violet?
- identifies neural structure in the brain and spinal cord tissue
- can detect damage and lesions.
- can monitor NEURODEGENERATION in animals.
- dark colour = high density of cells. MORE CELLS = takes up more stain.
CANNOT use on humans, unless POST MORTEM - but not a good choice anyway, because it’s not that specific.
Eg: Chemically lesion animal brain areas, then use this stain to see the extent of damage that was caused - as a manipulation check.
very good in animals.
What is golgi stain used for?
- Stains the ENTIRE NEURON, including dendrites and axon and CONNECTIONS between neurons can be seen - eg. uniploar, bi polar.
- complex networking structure can be investigated
- not good for detecting cell densities, bc not ALL neurons will stain
eg. detecting the effects of childhood neglect on neural connections, and growth of dendrites…
What is Luxol Fast Blue used for?
- stains Myelin
can look at enviro influences on myelin
What is amyloid stain used for?
detects amyloid deposits in the tissue.
- Alzheimer’s
- this stain isn’t specific for alzheimer’s disease
- amyloid is in extracellular space for AD
What is immunohistochemistry?
- Use if you know exactly what causes a condition - what specific PROTEIN you’re looking for.
- very SPECIFIC method.
- can combine with other stains.
- can look for AMYLOID PROTEIN
- is good because you can know that it’s not due to artefacts.
- Cannot let any of the proteins denature!! - use very GENTLE tissue preparation.
- If you let it denature, you will prevent the ANTIBODIES from binding to the ANTIGENS (proteins)
- PRODUCE antibodies that specific bind to your antigen.
- Can use high quality fluroescent dyes.
- very EXPENSIVE and SENSITIVE and SPECIFIC
- can look for multiple proteins at once, just attach different fluorescent markers to each
What is an example for something you’d use immunohistochemistry for?
To investigate Huntington’s disease.
- Look for cell loss - with other stains- can monitor the progression of the condition.
- Then specifically, (Especially in early stages before cell
loss, look for the protein that causes the condition
using immunohistochemistry.
Cell Analysis techniques?
Flow Cytometry Fluorescence microplate assays Cell counting Small animal in vivo imaging Super-resolution microscopy
Why is cell analysis good?
- Cells are the starting point of a myriad of biochemical and physical processes of life
- Can allow us to understand, predict and ultimately influence factors that underlie cell health, proliferation, function and death.
- good, specific, but not in living humans. only post mortem.
SO can’t monitor disease
—> CANCER! - when cells completely lose their identity.
What is small animal in vivo imaging?
- Used to investigate effects of drugs or movement of small molecules in the body of small animals.
- label the drug with flurosence so we can see it move/migrate from injection site and if it crosses BBB
- cannot see the EFFECT of the drug.
- also good to investigate DOSAGE of a drug - see how fast it exists the body.
- expensive machine.
ethics - good for ethics because we can keep an animal for longer. flush the drug do another trial.
What is super-resolution microscopy?
- investigates SYNAPTIC CONNECTIONS, different PROTEINS inside cell body, organelles, nuclear membranes…
- can look at sub-organelle level.
- info about fine structure of cells
eg. mitochondria - site for cellular respiration - problems with mitochondria = neurodegeneration
- must use VERY specific stains
- Pretty much anything can be visualised.
- very expensive type of microscopy.
What are enzyme immunoassays?
- Enzyme immunoassays developed for the QUANTITATIVE determination of proteins (antibodies or antigenes) in a biological sample.
- tissue extract, blood, CSF, Saliva, other Body Fluids.
- originally used to identify antibodies in blood
- need to think about which bio sample you want to use. eg. saliva is the easiest because doesn’t cause stress in participant –> might change levels of cortisol etc
What is the central dogma?
- The central dogma of molecular biology explains that DNA codes for RNA, which codes for proteins.
- Just having the gene for a protein (eg. mutated gene) doesn’t mean you will have the protein, this is why it’s important to look for the gene.
- So it’s important to investigate protein on top of the DNA
- DNA –> RNA –> PROTEIN
- DNA can’t leave nucleus, but RNA will be in the cytosol.
What is DNA Electrophoresis?
- identifies, quantifies and purifies nucleic acid FRAGMENTS - use with DNA, RNA and PROTEINS
- Samples are loaded into wells of agarose or acrylamide gel, and subjected to an electric field.
- -vely charged nucleid acid will move towards positive electrode.
- shorter fragments = travel more rapidly
- longest fragments = stays closest to origin
if you know the size of the gene fragment you’re looking for, you can take it out and then further investigate - the different bLOTS
what is southern blot?
- analysis for DNA identity, size and abundance
- do this after eletrophoresis, then transfer to a membrane, hybridise with a labeled sequence-specific probe, washing and finally detection of labelled DNA bands.
- design a probe to attach to the mutated part of the DNA - if it is not mutated, it will not bind - and thus you can visualise if there is a mutation in the gene.
- because the probe must be complimentary, it won’t bind it anything else - specific!!
What is Northern Blot?
- reveals information about RNA identity, size and abundance.
- Also after electrophoresis.
- allows for deeper understanding of gene EXPRESSION - good to identify if a protein is over or under expressed, but even if you have RNA it doesnt mean it’s expressed..
- difficult bc RNA is very sensitive - it isn’t meant to stay in the body for long
What is the Western Blot?
- Detects specific proteins in a sample of tissue homogenate or extract.
- gel electrophoresis separates the proteins by their length
- separated proteins are transferred to a membrane, where proteins are stained on the membrane using specific labelled antibodies.
what is PCR or RT-PCR used for?
Use this when you have very little tissue sample, or even a SINGLE CELL - eg. IVF - amplify then identify the mutation - can be used to prevent.
1 –> 2 –> 4
doubles it.
exponential.
What is epigenetics?
- Factors other than genotype that produces changes in a phenotype –> epigenetic change.
- two important processes that produce reversible changes in gene expression - HISTONE MODIFICATION, DNA METHYLATION.
What is histone modification?
Histone acetylation results in loose packing of nucleosomes.
–> loose = more expression
What is DNA methylation?
Causes nucleosomes to pack tightly together.
Transcription factors will not be able to bind to the DNA and genes wont be expressed
