Neuroscience Research Methods 1 Flashcards
Give four techniques used to perform functional research in neuroscience. (4)
- Microscopy
- Electrophysiology
- Behavioural testing
- Imaging
What is the difference between a naive animal and an animal model in the context of neuroscience research? (2)
Naive animal - no changes, used to understand normal function
Animal model - idealised or modified to represent a disease or physiological condition
A researcher is looking at hippocampal function. Her research question can be answered using a mouse model, but she would prefer to use a sheep because it would be better applied to humans. Is this possible for her to do? Why? (2)
No - the lowest sentient being possible should always be used
Give five advantages and one disadvantage of using drosophila/zebrafish models in neuroscience research. (6)
ADVANTAGES:
- Less complex nervous system
- Cheap
- Quick
- No licence
- Can answer fundamental questions
DISADVANTAGE:
- Harder to apply to humans
Give three advantages and one disadvantage of using mouse/rat (rodent) models in neuroscience research. (4)
ADVANTAGES:
- Mammalian nervous system
- Cheaper than larger mammals
- Quicker than larger mammals
DISADVANTAGE:
- Do not have folded cortex
Which types of rodent models are more suited to mice, and which to rats in neuroscience research? (2)
- Mice commonly used for genetic models
- Rats easier to train and larger
In which two ways are sheep brains similar to human brains (in the context of neuroscience research)? (2)
- Folded cortex
- Dura mater
Sheep brains contain a dura mater.
How can this fact be exploited when using sheep models in neuroscience research? (1)
Solid tentorium means that they can be used to investigate intracranial pressure.
Give three ways that animal models can be produced for neuroscience research. (3)
i.e. how can we manipulate animals to show features of a disease?
- Genetic modification
- Chemical/pharmacological
- Surgery
Give an example of a genetic modification being used to create an animal model for use in neuroscience research. (1)
Inserting CAG repeats into Huntingtin gene
Give an example of a chemical/pharmacological modification being used to create an animal model for use in neuroscience research. (1)
Inducing cell death of dopaminergic neurones using MPTP for Parkinson’s disease
Give an example of a surgical modification being used to create an animal model for use in neuroscience research. (1)
Occlusion of MCA with monofilament for stroke
Describe how a lesion-based animal model would be produced for use in neuroscience research.
Which category/categories does this model come under in terms of genetic/chemical/surgery? (2)
Create a lesion in a specific part of the brain using chemicals.
Comes under surgical and chemical/pharmacological categories.
Give four licences that must be obtained before beginning a research project involving the use of animals. (4)
- Establishment licence (PCD)
- AWERB (authorises need for project)
- Project licence (need for each experimental technique)
- Personal licence
In stereotactic neurosurgery, define bregma. (1)
Point where coronal and sagittal sutures intersect.
In stereotactic neurosurgery, what value is given to bregma. (1)
0
In stereotactic neurosurgery, define lambda. (1)
Point where the sagittal and lambdoid sutures intersect.
Describe the coordinate values moving anterior to bregma in stereotactic neurosurgery. (1)
Positive
Describe the coordinate values moving posterior to bregma in stereotactic neurosurgery. (1)
Negative
Describe the coordinate values moving lateral to bregma in stereotactic neurosurgery. (1)
Positive
Describe the coordinate values moving ventral to bregma in stereotactic neurosurgery. (1)
More positive moving downwards.
Define ‘cell culture’. (1)
The growth of cells from an animal or plant in an artificial, controlled environment.
Give two challenges faced when culturing neurones. (2)
- Mature neurones do not undergo cell division
- Neurones have complex morphologies and connections
Describe three considerations to take into account regarding the environment when performing cell cultures. (3)
- Components needed for cellular metabolism
- Need for incubators
- Avoidance of contamination
Give three different neuronal cell models that can be used for cell culture. (3)
- Immortalised cell lines (neuroblastoma)
- Primary neurones
- Induced pluripotent stem cells
Give two advantages and two disadvantages of using neuroblastoma immortalised cell lines in cell cultures. (4)
ADVANTAGES:
- Continuous cell division
- Can use a human model
DISADVANTAGES:
- Differentiated state does not produce a typical neuronal structure
- Cannot be used in compartmentalised systems
Describe how primary neurones would be obtained to be cultured and used in neuroscience research. (1)
Cells derived from embryonic neuronal tissue
Give three advantages and one disadvantage of using primary neurones in culture for neuroscience research. (4)
ADVANTAGES:
- More typical neuronal morphology
- Can develop neuronal networks in vitro
- Can adapt to cultures which grow multiple different cells
DISADVANTAGE:
- No more cell division
Describe what is meant by an ‘induced pluripotent stem cell’, as applied to neuronal culture and neuroscience research. (2)
- Adult somatic cells which have been genetically reprogrammed to a state similar to embryonic stem cells.
- Cells then stimulated to differentiate into neurones.
Give one advantage and two disadvantages of using iPSCs to culture neurones and carry out neuroscience research. (3)
ADVANTAGE:
- Can create a human model which accurately represents a specific disease state
DISADVANTAGES:
- Expensive
- Variability in results (due to differences in human cells)
Describe why neuronal cell cultures cannot just be grown as a sheet of cells in a petri dish.
Why do we need to use special types of neuronal cell culture systems? (2)
Neurones have complex morphologies
and different culture platforms may be required for different parts of the cell.
(eg, cell bodies, and axons, and dendrites may need different conditions to grow)
Give three types of cell culture system which may be ideal to grow neurones. (3)
- Microfluidic cultures
- 3D systems
- Brain organoids (which are another type of 3D system)
Describe the principles behind the microfluidic cell culture technique, and explain why it might be an ideal environment to culture neurones. (2)
- A compartmentalised system which is used to culture primary neurones.
- Ideal to grow neurones because different drugs/reagents can be added to each side (ie. dendrites vs axons)
Describe the principles behind the 3D cell culture technique, and explain why it might be an ideal environment to culture neurones. (2)
Different cell types cultured on a model of extracellular matrix.
May be ideal to grow neurones because it attempts to mimic the anatomical structure of the nervous system.
Give two disadvantages of the 3D cell culture technique. (2)
- Expensive
- Powerful microscopy techniques required for visualisation
Describe the principles behind the brain organoid cell culture technique. (1)
3D culture produced of neural cell aggregates.
(Mini brain containing neurones and glia)
Give an advantage and disadvantage of the brain organoid cell culture system. (2)
ADVANTAGE:
- More accurate model for brain development
DISADVANTAGE:
- More difficult to view individual cells
Briefly name the steps involved in the trizol method of RNA isolation. (4)
- Lyse cells
- Separate RNA via mixing or shaking
- Centrifuge
- Precipitate RNA
When isolating RNA using the trizol method, what reagent is used to lyse/homogenise the tissue? (1)
Trizol
When isolating RNA using the trizol method, how can the trizol reagent help preserve the integrity of the RNA? (1)
Inhibiting RNase enzymes
When isolating RNA using the trizol method, which reagent is used to help the RNA separate from the DNA/proteins in the sample? (1)
Chloroform
When isolating RNA using the trizol method, which layer produced after centrifugation will contain the RNA? (1)
Top
When isolating RNA using the trizol method, what reagent is added to precipitate RNA molecules after centrifugation? (1)
Ethanol
Briefly name the steps involved in DNA isolation. (4)
- Lyse and homogenise cells
- Treat with proteases
- Centrifugation
- Precipitate DNA
When isolating DNA, why do cells have to be lysed/homogenised? (1)
To dissolve plasma and nuclear membranes and release cell contents.
When isolating DNA, why is the sample treated with proteases? (1)
To destroy proteins such as histones and allow DNA to unwind
When isolating DNA, what reagent is used after centrifugation to precipitate the DNA? (1)
Ethanol
Give two uses of PCR. (2)
- Measure gene/RNA expression
- Manipulate the genome/transcriptome
Give four techniques which are commonly paired with PCR in neuroscience research. (4)
- QPCR
- DNA Microarrays
- In situ hybridisation
- Sequencing
Name the enzyme usually used in PCR.
Where does this enzyme come from, and why is it ideal for use in PCR? (3)
Taq polymerase (form of DNA polymerase)
First discovered in bacteria
Ideal because it is very heat resistant (optimum temp = 75-80C)
Briefly describe the fundamental principle of PCR. (2)
Uses a temperature cycle
to theoretically produce infinite copies of a small sample of DNA.
Describe the three steps in PCR, and the rough temperatures that they are carried out at. (3)
Denaturation (92-94)
Annealing (50-70)
Elongation (~72)
Describe what is meant by ‘hot start PCR’, and describe why it is used. (3)
- Specific form of Taq polymerase only active at high temperatures
- So amplification can only start once temperature is raised and first denaturation has occurred
- Which can help unwanted/random amplifications and primer dimers forming during PCR setup at room temperature
Is PCR performed on DNA or RNA?
Give two reasons why? (3)
DNA
- RNA too unstable
- Taq polymerase does not work on RNA, but cannot use RNA polymerase as it would not withstand heat
If PCR can only be performed on DNA, how can RNA expression for a specific gene be measured, as the original sample will consist of RNA? (1)
Reverse transcription to form DNA from RNA
Give five ‘ingredients’ that must be added together in an eppendorf when performing PCR. (5)
- 2x primers (forward and reverse)
- Nucleotides
- Taq polymerase
- DNA template of interest
- Buffer
Describe the denaturation step in PCR. (2)
High temperature causes hydrogen bonds between DNA strands to break.
DNA separates into single strands.
Describe the annealing step of PCR. (1)
The primer binds to complementary sequence on DNA.
Describe what affects the temperature that annealing must be carried out at when performing PCR. (2)
Base composition and length of primer.
Also, temp must be ideal for both forward and reverse primers.
Describe (in reasonable detail) the elongation step in PCR. (3)
- Taq polymerase binds to 3’ end of DNA
- Polymerase copies complementary sequence
- New DNA strand formed 5’ to 3’
Describe how a sample of material may be visualised after performing PCR.
How would the sample look when it comes out of the thermocycler? (3)
Run on agarose gel.
Visualise using UV light.
(Gel electrophoresis)
- The sample appears as a transparent liquid
What is QPCR used for? (1)
Quantify the amount of RNA (and therefore specific genes/gene expression) in a sample.
Describe simply the basic principles surrounding QPCR. (4)
- DNA detected via fluorescent probe
- Detection threshold set (intensity of fluorescence)
- More DNA (and therefore RNA) in original sample = less cycles to reach threshold
- Cycles to threshold can be compared to known control
What is meant by the term Ct, in QPCR? (1)
Cycles to threshold - higher Ct means less RNA in original sample
Describe how you can work out the fold-change in RNA expression by looking at the Ct of two samples in QPCR.
Every Ct difference = changed by a factor of 2
Eg - Ct 17 and Ct19 means that expression will have changed by a factor of 4.
Describe the three phases of QPCR, in terms of how fast the reaction occurs.
In which phase of the reaction should the detection threshold be set in? (4)
- First exponential phase
- Then linear phase (contains detection threshold)
- Then plateau phase
Why does a PCR/QPCR reaction eventually plateau? (2)
May be due to denaturation of Taq polymerase or depletion of primers.
However noone really knows.
What factor limits the linear phase of a PCR/QPCR reaction? (1)
Number of Taq polymerase molecules
What is DNA called if it has been produced from an RNA sample via reverse transcription? (1)
cDNA
Name two methods of QPCR. (2)
i.e. 2 ways to add fluorescent probes to the DNA for detection
SYBRGreen
TAQMan
Give a brief overview of how SYBRGreen QPCR works to produce a signal. (3)
- Add SYBRGreen molecules to solution undergoing PCR
- Molecules intercalate nonspecifically into dsDNA molecules
- When molecules are incorporated into dsDNA they fluoresce
Give two advantages and one disadvantage of the SYBRGreen method of QPCR as opposed to TAQMan. (3)
ADVANTAGES:
- Cheaper
- Easier
(regular, non-modified primers can be used)
DISADVANTAGE:
- SYBRGreen binding is nonspecific so you may be picking up DNA contaminants as well as gene of interest
After QPCR with SYBRGreen has been carried out, give another step of the experiment which is performed. (1)
Melt analysis
Why is melt analysis performed after SYBRGreen QPCR? (1)
To determine whether a single product or multiple products have been obtained.
Describe the principles behind melt analysis for SYBRGreen QPCR. (4)
- Temp slowly increased
- dsDNA will denature and stop fluorescing
- However different products will denature at different temperatures
- So if >1 product produced, multiple different melting profiles will be observed
Describe the principles behind the TAQMan method of QPCR. (3)
- In addition to regular primers, a probe primer is added (attached to fluorescent probe)
- Probe primer has base sequence complementary to gene of interest (but different from regular primers)
- When Taq polymerase reaches probe primer during elongation, it digests fluorescent molecule, which begins to fluoresce
Give one advantage and one disadvantage of TAQMan QPCR compared to SYBRGreen. (2)
ADVANTAGE:
- High specificity for gene of interest (contamination will not affect signal)
DISADVANTAGE:
- Requires special and expensive primer probes
What are DNA microarrays used for in neuroscience research? (1)
Measure gene expression/amount of RNA/amount of DNA
Describe the principles behind a DNA microarray. (4)
- Glass or silicone slide containing spots filled with ssDNA probes of known base sequence
- Sample of interest fluorescently labelled and incubated with microarray
- Sample binds to complementary DNA spot and unbound sample washed away
- Measure fluorescence of each spot and compare relative expression between samples
Describe how a sample of interest DNA can be compared to a sample of control DNA with known expression using a spotted DNA array.
Describe how the results would be interpreted. (5)
- Control and sample labelled with different fluorescent colours (eg. control red and sample green)
- Control and sample hybridised to same spot on DNA array
- Colour closer to red = more control
- Colour closer to green = more sample
- Yellow = equal
Give a disadvantage of using spotted DNA arrays to measure gene expression. (1)
Can only compare results from the 2 samples hybridised to the same spot.
Describe how affymetrix gene chips work to compare expression between different DNA samples. (3)
- Gene chips contains many short probes for many different genes
- Samples of interest loaded onto gene chip
- Signal of bound DNA measured and gene expression can be compared between many samples
Describe an advantage of affymetrix gene chips compared to spotted DNA arrays for comparing levels of gene expression. (2)
Can compare many control/treatment samples
because affymetrix oligonucleotides normalise expression between chips.
Describe how DNA microarrays can be used to measure gene expression, given that DNA is the same in all cells. (2)
Take an original RNA sample
and reverse transcription to make cDNA.
(More RNA in original sample = more DNA in test sample, then measure amount of DNA using microarray)
What is in-situ hybridisation used for in neuroscience research? (1)
Localising specific nucleic acid targets within tissues, cells, and chromosomes.
True or false? (1)
In situ hybridisation is good for measuring gene expression in a tissue sample.
False - it is used to see WHERE a gene or nucleotide sequence is expressed
Briefly describe how a tissue or cell sample would be prepared prior to in-situ hybridisation. (3)
- Samples preserved (fixed/frozen)
- Thinly sliced
- Attached to glass slide for support
Describe how targets are localised and detected in in-situ hybridisation. (4)
Complementary probes produced
Labelled with fluorescence or radioactivity
Probes hybridised with tissue samples, and they bind to complementary DNA/RNA
Fluorescence or radioactivity then measured in sample
Describe the main difference between in-situ hybridisation and immunohistochemistry. (2)
In-situ hybridisation identifies nucleotide targets.
Immunohistochemistry identifies protein targets.
Briefly describe how sanger sequencing is carried out. (4)
- PCR carried out with both normal base pairs and chain-terminating base pairs
- Chain-terminating bases labelled a different colour or carried out using only one base at a time
- Fragments separated by size (eg. electrophoresis)
- Colour of each fragment indicates last base in sequence
Describe the principles behind pyrosequencing. (3)
- Based on the principle that each time a base pair is added, pyrophosphate is released
- Pyrophosphate undergoes chain of events, including luciferase reaction, to produce light
- Each possible base pair is added separately, and if light is emitted, it confirms that the base has bound and is the next in the sequence
Briefly describe emulsion PCR - a technique that is used in second generation sequencing. (4)
- One DNA strand attached to solid base (eg. microbead)
- DNA strand and base contained in droplet (eg. aqueous droplet in an oil emulsion)
- Multiple droplets can then be put in one mixture as they are separated
- They can undergo PCR at the same time
Define ‘recombinant DNA technology’. (2)
The creation of new DNA molecules,
or the transfer of genetic information to/from an organism.
Name the steps required for making recombinant DNA. (6)
- Isolate DNA
- Cut DNA
- Join DNA
- Introduce DNA into host cell
- Allow cells to multiply and select clones containing target DNA
- Express target DNA
Give three enzymes that may be used for recombinant DNA technology, and briefly describe their role. (6)
- Restriction endonuclease (cleave DNA at specific sites)
- Ligase (join bits of DNA together)
- Phosphorylase (turn genes on/off)
Why is mechanical shearing NOT commonly used to cut DNA molecules during recombinant DNA technology? (1)
Mechanical shearing only cuts the DNA at random locations.
In recombinant DNA technology, describe how restriction endonucleases are used to cut the DNA molecule. (3)
DNA cut at a unique site recognised by the enzyme.
Sometimes 2 different enzymes are used on the same piece of DNA
to allow the right ends to fit into the vector.
In recombinant DNA technology, how are the two (or more) pieces of cut DNA joined together? (1)
Using ligases
In recombinant DNA technology, give another name for introducing the new DNA molecule into a host cell. (1)
Transfection
Name four methods of transfection used in recombinant DNA technology to insert a new piece of DNA into a cell. (4)
- Chemical transformation
- Electroporation
- Lipofectamine
- Injection
Describe how chemical transformation can be used to insert new DNA molecules into cells during recombinant DNA technology. (2)
DNA plasmids and cells are mixed
then exposed to heat shock treatment.
Describe how electroporation can be used to insert new DNA molecules into cells during recombinant DNA technology. (1)
Short high-voltage pulses used to break down cell membrane.
Describe how lipofectamine can be used to insert new DNA molecules into cells during recombinant DNA technology. (2)
Spheres of phospholipids containing DNA
are fused with the cell membrane.
Describe what is meant, and what is used as a vector in recombinant DNA technology. (2)
- Bacterial plasmid used
Vector is the DNA sequence into which cut section of DNA will be inserted to be carried into new cell or organism.
True or false? (1)
Recombinant DNA technology uses viruses to insert foreign DNA into a cell.
False - uses plasmids as a vector
What is the role of RNAi (RNA interference)? (1)
Turn genes off
What is the difference between RNAi and miRNA? (2)
RNAi is a way to manipulate cells.
miRNA occurs naturally in cells.
Briefly describe the steps in RNAi, not including the enzymes and proteins involved. (5)
- Synthetic precursor dsRNA enters cell
- dsRNA cut into short fragments
- Fragments split into passenger strand and guide strand
- Guide strand guides enzyme to target mRNA
- mRNA cleaved
In RNAi, name the enzyme which cuts the precursor dsRNA into short fragments. (1)
Dicer
In RNAi, name the protein complex which cleaves the target mRNA.
Name the enzyme associated with this complex and state its role. (3)
RNA-induced silencing complex (RISC)
The AGO2 enzyme is associated with RISC,
and it binds to the dsRNA fragments and splits them into guide and passenger strands.
In RNAi, after the dsRNA has been cleaved to form fragments by dicer, what is the RNA now referred to as? (1)
short interfering RNA (siRNA)
What is a viral vector, and what is it used for? (1)
Viral vectors are delivery systems used to introduce foreign genetic material into specific cells.
Describe the steps required to build a viral vector. (4)
- Design gene of interest
- Insert gene into shuttle vector (eg. plasmid)
- Insert gene/plasmid plus virus packaging mix into a eukaryotic cell
- Harvest the virus once it has been assembled inside the cell
Name four viral vectors which can potentially be used in neuroscience research. (4)
- Adeno Associated Virus (AAV)
- Retrovirus
- Adenovirus
- Herpes Virus
Give two neurological conditions which are currently being clinically investigated using viral vectors. (2)
- Parkinson’s
- Alzheimer’s
Name a viral vector which is commonly used to manipulate the genome in neuroscience research, but cannot be used clinically due to vector toxicity. (1)
Adenovirus
Define what is meant by a ‘transgenic animal’. (1)
An animal whose genome has been modified.
Give three general ways in which a transgenic animal’s genome can be modified. (3)
- Knock out (gene removed)
- Knock in (new gene inserted)
- Knock down (gene expression reduced)
Describe what is meant by…
a) inducible
b) constitutive
gene alterations. (2)
a) Activation/inactivation of a gene only in specific cells at specific times
b) Continuous on/off change to a gene
Give four ways that transgenic animals may be created. (4)
- DNA microinjection
- Transposons
- Lentiviral vectors
- Chimerae
Briefly describe the DNA microinjection technique for creating transgenic animals. (1)
- DNA directly introduced into cell via a microinjection
Briefly describe the transposon technique for creating transgenic animals. (1)
Using genes that can transport to a different location in the genome to transport the new gene
Briefly describe the lentiviral vector technique for creating transgenic animals. (1)
Using viral transduction to introduce the new DNA into a cell.
What is a chimeric animal? (1)
An animal containing cells with more than one genotype.
ie. an animal with genes from two or more different species
Describe simply how you could clone a transgenic animal. (3)
Egg donor from one animal with genetic material removed.
Genetic material from readily-produced transgenic cells inserted into enucleated eggs.
Inject cells into host to develop.
What is CRISPR Cas9 used for? (1)
Editing the genome at a precise location
How was CRISPR Cas9 first discovered? (1)
In a bacterial immune system
Describe the two components which make up CRISPR Cas9. (4)
Cas9 (DNA cutting enzyme)
Guide RNA (scaffolding sequence which binds to Cas9 plus a sequence which is complementary to DNA target)
Describe how CRISPR Cas9 works. (4)
- Short nucleotide sequence in DNA called PAM binds to Cas9
- PAM close to cleavage site so guide RNA can unwind complementary/target dsDNA
- Cas9 enzyme cleaves DNA and causes double strand break
- As cell tries to fix the cleaved DNA it can incorporate ‘false’ DNA which has been injected containing mutations/insertions/deletions
What is the importance of the CRISPR Cas9 technique making a double stranded break in the DNA of a cell? (2)
A double strand break would usually be fatal for a cell’s genetic material.
So the cell will always try to fix the cleaved DNA (and can therefore incorporate inserted DNA).
What is gel electrophoresis used for? (1)
Gel electrophoresis is used to separate DNA, RNA, or proteins by their molecular weight/size.
Describe the process and interpretation of the results for gel electrophoresis. (5)
- Load samples into wells/columns
- Current produced which moves samples (negative to positive)
- Smaller molecules move faster through the gel
- So when the reaction stops the molecules which have moved furthest are smaller
- Sample with known sizes can be run as a ladder for quantitative analysis
What would happen if a sample of organic material containing molecules of different charges was separated using gel electrophoresis? (2)
- Molecules separated by both size and charge
- Movement based on charge:mass ratio
Describe the gels which would be used in gel electrophoresis to separate:
a) DNA/RNA
b) proteins
Why do different gels have to be used?
(3)
a) agarose gel
b) polyacrylamide gel
Proteins too small to be run on an agarose gel.
Complete the passage regarding the separation of DNA fragments via gel electrophoresis. (3)
DNA fragments will move towards the …………………… electrode, because they are ………………….. charged due to the presence of …………………… groups.
Positive
Negatively
Phosphate
How are bands of DNA visualised after performing gel electrophoresis? (1)
Original samples mixed with fluorescent dye
What is an epitope? (1)
Specific area of an antigen where the antibody binds.
Briefly describe how a polyclonal antiserum would be produced for use in neuroscience research. (2)
- Inject antigen into mouse
- Isolate serum
Briefly describe how monoclonal antibodies would be produced for use in neuroscience research. (6)
Inject antigen into mouse.
Isolate immune cells which form antibodies.
Fuse immune cell with tumour cell to form hybridomas.
Hybridomas screened for production of desired antibody.
Antibody-producing hybridomas cloned.
Isolate antibodies.
Describe the difference between monoclonal antibodies and polyclonal antiserum. (2)
MONOCLONAL ANTIBODIES bind specifically to just one epitope of an antigen.
POLYCLONAL ANTISERUM contains a number of different antibodies, so all epitopes of an antigen will end up being bound.
Give two advantages and one disadvantage of using monoclonal antibodies in neuroscience research, as opposed to polyclonal antiserum. (3)
ADVANTAGES:
- Very specific - less noise in experiments
- Unlimited supply - hybridoma cells continue replicating
DISADVANTAGES:
- Expensive
Give one advantage and one disadvantage of using polyclonal antiserum in neuroscience research, as opposed to monoclonal antibodies. (2)
ADVANTAGE:
- cheaper
DISADVANTAGE:
- binding less specific - produces more background noise
What is ELISA used for? (1)
Detecting antibodies or antigens present in a liquid (eg. serum) or homogenised tissue.
Briefly, how can levels of an antibody or antigen be quantified in ELISA? (1)
Change in colour
Briefly describe how to perform indirect ELISA. (4)
Well coated with antigen (complementary to antibody of interest)
Sample containing antibody of interest added to well
Second antibody (against first antibody), conjugated with enzyme, is added
Substrate added and will change colour if second antibody has bound
*wash between each step
In indirect ELISA, describe the results. (2)
ie. what is measured, does darker colour mean more/less?
Measures antibodies of interest.
Darker colour means more antibodies present in original sample.
Briefly describe how to perform sandwich ELISA. (4)
Antibody (complementary to antigen of interest) attached to well.
Sample containing antigen of interest added.
Secondary antibody (enzyme conjugated) added.
Substrate added and changes colour.
*Wash in between steps
In sandwich ELISA, describe the results. (2)
ie. what is measured, does darker colour mean more/less?
Measures antigens of interest.
Darker colour = more antigens present in sample
Briefly describe how to perform competitive ELISA. (5)
Excess of antibody incubated with antigen of interest.
Well coated with antigen which also binds to antibody.
Add antigen-antibody complexes to well (only free antibodies not already occupied will bind to antigens in well)
Enzyme-conjugated secondary antibodies added.
Substrate added and changes colour.
*Wash between each step
In competitive ELISA, describe the results. (2)
ie. what is measured, does darker colour mean more/less?
Technique measures antigens.
The lower the signal, the more antigens present in the original sample
(more primary antibodies occupied by antigens of interest, so less bind to well)
Describe what immunoprecipitation is and what it is used for. (2)
Specific separation (by precipitation) of molecules in a liquid or homogenised tissue
using antibodies.
What kinds of molecules can be separated from a mixture using immunoprecipitation? (3)
- Proteins
- DNA
- Post-translational modifications
Briefly describe the principles of how immunoprecipitation is performed. (3)
- Specific antibodies modified to allow easy separation
- Antibodies added to liquid/homogenate (and bind to targets)
- Antibodies and bound products precipitated to separate them from rest of molecules
Describe two ways in which antibodies are modified for easy separation and separated from the sample in immunoprecipitation. (4)
ANTIBODIES LINKED TO AGAROSE BEADS
- agarose beads give Abs higher density so can use centrifugation
ANTIBODIES LINKED TO MAGNETIC BEADS
- magnet can be used (outside tube) to attract and remove antibodies bound to magnetic beads
