Lecture 2: Sample Preparation and Extraction 1 Flashcards
What problems do we encounter that highlight the need for sample preparation?
Most biological/clinical samples are heterogeneous/mixtures (e.g. blood/ biopsies/ urine/ swabs).
Measurements usually target one analyte at a time (often found in trace amounts).
Difficulties in detection.
Direct analysis is detrimental to analytical instruments.
What solutions do we have surrounding the problems highlighting the need for sample preparation?
Reduce sample complexity.
Target the analyte of interest from the mixture using sample preparation methods.
Exploit the molecular characteristics of the analyte (Molecular size, polarity, functional groups, solubility, etc..)
Convert to a (more) detectable form and ensure analytical tool compatibility (e.g. chromatography, imaging, etc).
What is dilution?
Reducing concentration so it’s more suitable for analysis
What do you need to ensure for dilution and why?
Ensure Instrument compatibility: Limited linear calibration/detection range (when you measure something, it must be in its dynamic range – not too low, not too high).
Examples of when dilution is used
Used in calibration curves for quantification or cell counting.
What is homogenisation/dissociation used for?
For heterogenous samples (e.g. solid/soft tissues).
- Isolation of intact cells from tissues.
- Lyse cells to recover subcellular components (nucleic acids/proteins).
What methods can be used for homogenisation/dissociation?
Mechanical shear or with surfactants/enzyme-assist.
Disrupt membranes by ultrasonication (high-frequency sound) (32-38kHz)
Freeze-thaw cycling (water forms crystals that punch holes in the cells and make cells expand when frozen which breaks them)
What is tissue sectioning?
Slices of tissue (biopsies) for microscopy done with microtome or cryostat.
When is a microtome used for tissue sectioning?
Microtome: formalin fixed paraffin-embedded tissue (room temp)
When is a cryostat used for tissue sectioning?
Cryostat: fresh frozen tissues to monitor real-time biological processes (fewer artefacts from formalin processing and maintains structures e.g. metabolites).
What can tissue sectioning preparation lead to downstream?
- Tissue staining – morphological features
- Immunohistochemistry – protein biomarker
- Mass spec imaging – MALDI-MS
- In-situ hybridisation (ISH) – expression analysis
What is fixation for?
After cryo-sectioning to preserve samples in ‘life-like state’ at a ‘fixed’ point in time – tissue sample resilience (ease of transport/storage).
Stops degradation/degeneration process (autolysis) and prevents diffusion of soluble substances.
Required for scanning and transmission EM.
What are the methods of fixation?
chemical fixation
physical fixation
perfusion
How does chemical fixation work?
ethanol/ formaldehyde/ glutaraldehyde (decreases solubility and allows protein cross-linking).
How does physical fixation work?
heating to 37-45 degrees C to increase diffusion rates.
How does perfusion work in fixation?
Distributes buffers/fixative agents via circulatory system - remove interferences from blood.
What is the purpose of antigen retrieval?
Treatment of tissue sections to unmask/retrieve antigens masked/altered during fixation – breakage of cross-links or restoration of accessibility of epitopes (binding sites for antibody receptors).
Crucial before immunohistochemistry (IHC), immunofluorescence (IF) and in situ hybridisation (ISH) techniques.
What are the types of antigen retrieval?
Heat-induced antigen retrieval
Proteolytic-induced antigen retrieval
Explain proteolytic-induced antigen retrieval
Proteolytic-induced antigen retrieval (enhance antibody access – causes some tissue degradation).
- Pepsin, proteinase K, and trypsin proteases used at neutral pH and 37 degrees C.
Explain heat-induced antigen retrieval
Heat-induced antigen retrieval (denature bridges).
- Citrate pH4.5-6 retrieval buffer for general epitopes & Tris-EDTA pH9 buffer for nuclear antigens (transcription factors inside cells).
- High temperature (95 degrees C) water bath or pulsed microwave treatment.
What is the purpose of precipitation?
To reduce analyte solubility (crashing/come out of solution).
What are the methods of precipitation?
salts
trichloroacetic acid (pH change)
solvents
immunoprecipitation
temperature
Explain the use of salts in precipitation
Salts: protein aggregation in high-conc. ammonium sulphate (removes water – salt draws the water out).
Explain the use of Trichloroacetic acid (pH change) in precipitation
Trichloroacetic acid (pH change): denaturing of tissues/proteins, disrupts hydrogen bonding but leaves peptide bonds intact.
Explain the use of solvents in precipitation
Solvents: ethanol, acetone, methanol/chloroform lowers dielectric constant (decrease solubility – lowers water’s ability to solubilise or selective dissolution).
Explain the use of immunoprecipitation in precipitation
Immunoprecipitation: antibodies to ‘fish out’ target analytes (antibodies bind to analytes - makes them insoluble).
Explain the use of temperature in precipitation
Temperature: cold lowers solubility (degradation hampered in lower temperatures).
Explain filtration/dialysis as a sample preparation technique
Size inclusion/exclusion:
- Reverse osmosis (0.0001μm – 0.001μm)
- Ultrafiltration (0.01μm – 0.1μm)
- Microfiltration (0.1μm - 1μm)
- Clarification (10μm - 100μm)
Filters:
- Paper discs (remove large/insoluble particles), membranes (sterile fixation – filters out pathogens), glass fibres
- Gravity, vacuum, high pressure
- Centrifuge filtration (spin columns – no adhesion, just a filter).
What is the purpose of Size-exclusion chromatography (SEC)?
Sort/separate analytes (filtration) by size, aggregation, conformation, and hydrodynamic radius (e.g. proteins, polymers and nanoparticles).
Describe the columns in SEC and how this separates analytes.
Columns:
- Porous non-adsorbent stationary phase resin (different fractionation range based on pore size).
- Elution by isocratic mobile phase
- Detection by UV-Vis (light scattering)
o 280nm – proteins
o 260nm – nucleic acids
- Fraction collection
Smaller analytes take longer to pass through the column (smaller particles have a longer path unlike the bigger ones which go straight through) - good for purification.
What is the purpose of centrifugation?
Separation by differences in density, buoyancy, sedimentation rate (denser goes to bottom).
- (non) Cellular components of blood (Ficoll-paque, histopaque, lymphoprep, percoll).
- Isolating sub-cellular components of lysed cells
(Ultracentrifugation (gravity 20000<)
What is the purpose of quenching?
Halt/freeze chemical reaction at specific timepoint (Enzyme kinetics, chemical synthesis, analysis of intermediate product, monitoring reaction states).
By changing temperature or reaction mixture (with acids, bases, solvents) rapidly to preserve the substrates and products (by stopping the enzyme).
‘stop solutions’ in ELISAs
Why is derivatisation an important technique?
mass spec most commonly used to analyse so analytes must be ionised and derivatisation helps with this
Explain derivatisation
Labelling/tagging – alter chemical structure etc. to increase detectability, identification, ionisation efficiency, sensitivity, selectivity, stability (gives constant charge (ionised) therefore always polar).
Enable high-throughput analysis:
- Multiplexing – derivatives of reagent taken (short time/lots of samples) with different hydrazine tags that have different masses.
- Isobaric mass-tags in proteomics (<16 plex).
in GC-MS analysis – increase volatility, thermal stability, and aid separation (needs to be non-polar to decrease the boiling point to become gas easily – to be volatile)
- Trimethylsilylation (polar tagging) of oestrogens (estrone and estradiol)
What is the use for perfusion in medical imaging?
In nuclear/radiation medicine to assess blood flow, organ function, metabolic activity, etc.
