Method Flashcards
How does cell passaging occur?
Set up the protocol in a tissue culture hood - so everything can be done in a sterile environment
Sterilise by spraying ethanol
Then look under the microscope to check the cells for contamination/infection
Remove the culture medium using a sterile pipette tip
Wash the cells with PBS solution, inserting with a new pipette tip
Then discard the PBS
Replace pipette tip and add in trypsin EDTA solution
Leave for 10 minutes and allow the cells to detach
Check if the cells have detached under a microscope
Using a 5ml pipette tip, break up the clumps of cells by pipetting up and down
Add in culture medium to the cells
Take a sample and work out the cell density using a heamocytometer and counter
Based on the cell count determine the ratio the cells that need to be passaged
Remove the excess cell solution and top up with media to make up 10ml in the flask
Update passage number on flask
Where is the genetic material kept inside the fertilised zebrafish embryo?
Cell
The other areas - chorion, yolk
What is the mid-blastula transition?
When the embryo starts transcribing its own genome
Occurs at the 1000 cell stage
Until then the embryo used mRNAs deposited in the egg by the mother prior to fertilisation
What are the 3 layers formed during gastrulation?
Ectoderm (from epiblast), mesoderm, endoderm (hypoblast)
What are the developmental stages of the embryo?
Cleavage - 1st step Blastula - 2nd step Gastrula - 3rd step Segmentation - 4th step Organogenesis - 5th step
When does the heart start beating?
36-48 hpf
Can be as early 28hpf
When does the embryo start moving?
Before coming out of the chorion
List some other organisms can be used to understand human development
Mouse
Chick
Fruit fly
Nematode/frogs
What structures does the endoderm give rise to?
Gives rise to majority of digestive tract - entire epithelium
What structures does the ectoderm give rise to?
Nervous system + epidermis
What structures does the mesoderm give rise to?
Muscle cells + connective tissue
Movements during gastrulation
Epiboly
Involution
Convergent extension
Radial intercalation
What is the notochord?
Defining structure of the chordates, and has essential roles in vertebrate development
Major skeletal element of the developing embryo
What is the 3 tier licensing system?
Establishment licence - certificate of designation
Project licence - specific research/testing programme
Personal licence - specific individual/competency
When are the licences approved?
If benefit outweighs the cost
If there is no non-animal alternative
Minimum number of possible animals used
Using animals with the lowest sensitivity to pain as possible
Pain is minimised
Research premises have necessary facilities to care for animals
What is the local ethical review?
Committee of scientists and lay people, review and justify the activity and use of animals
Done for every research/testing activity
What is required to carry out animal research?
Licences and approval from LEC
What are the 3 Rs?
Replacement - use of non-animal testing methods/alternative techniques
Refinement - welfare, improve procedures, better housing
Reduction - minimum number of animals, fewer animals, more information
What year was the Animal Scientific Procedure Act brought in?
1986
What does ASPA 1986 do?
Regulates use of protected animals in experimental or other scientific procedure which may cause pain, suffering, distress or lasting harm to the animal.
Which animals are considered by ASPA?
Any living vertebrae (other than humans) and any living cephalopods
What does ASPA ensure? (Animal Scientific Procedures Act)
Animals are cared with the best standards of animal husbandry
Home office inspection system in place to ensure rules are not violated
What is the central dogma?
DNA–>RNA–>Protein
Which sites regulate the expression of a gene?
Regulatory regions
eg. promotor
What direction is a sense strand?
5’ to 3’
Describe transcription
Transcription = unwinding DNA double helix, exposes anti-sense strand
Allows RNA pol to bind
Transcribe RNA in 5’->3’ direction (using anti-sense strand as a template)
generates RNA in sense orientation (5’ left, 3’ right)
When does transcription occur?
Genomic DNA transcribed in cells where the regulatory regions are activated
What happens after RNA is transcribed?
Processed, introns removed, 5’end modified, 3’ poly A tail = mature mRNA
Exported into cytoplasm
Serves as template for translation
Briefly describe the process of translation
mRNA positioned within the ribosome (between 2 subunits)
tRNA with amino acid attached, recognises codon, binds and allows polypeptide chain to grow
What do expression pattern of genes allow?
Give ideas about function of gene
How can the expression pattern of genes be visualised?
Staining techniques
In-situ hybridisation - localisation of mRNA within tissue
Immunostaining
What is in-situ hybridisation?
Looks at mRNA
Recognised using a specific RNA probe
Probe has DIG (digoxigenin)
Probe hybridised overnight to embryo
PBS washing to remove anything not specifically bound to mRNA
Incubate embryo with Ab recognises + binds to DIG label on probe
Ab has enzyme attached (Alkaline phosphatase)
Wash again
Add substrate for AP (BCIP/NBT) undergoes a colour reaction turns it into a coloured precipitate (dark purple)
Gives staining visible in regions contain mRNA
What does in-situ hybridisation show?
Gives staining visible in regions contain mRNA
shows how much mRNA is present - approx
How else can in-situ hybridisation be carried out (without enzyme linked Ab system)?
Fluorescently label Ab, detect by fluorescence
= FISH
What is immunostaining?
Looks at gene expression by looking at protein
Describe immunostaining
Generate Ab that recognises Ag (a protein in cell/tissue)
Amplify the signal using a secondary Ab which recognises the primary Ab (Ig domain)
NB. secondary Ab is generic for Ig
primary Ab is specific to the protein itself
2nd Ab can have AP attached - will change colour when add a substrate
Can also be fluorescent
Can combine different Ab and different colours on one specimen + look at 2 or more proteins at a time
What colours are fluorescein and rhodamine?
Fluorescein = green Rhodamine = red
How is a probe, to recognise mRNA in tissue, made?
Make cDNA from mRNA using reverse transcription to generate ssDNA
Then make dsDNA + amplify by PCR = cDNA
c DNA (ds) used to generate anti-sense RNA complimentary to sense mRNA that want to detect present within the sample
Antisense RNA has DIG attached (probe)
Incubate with embryos
Able to detect where probe binds to specifically + mRNA accumulation within the cell
Probe production in more detail
mRNA want to detect made by the cell from DNA in cell
mRNA processed by cell + has poly-A tail attached
This is the sense strand of RNA
cDNA made through reverse transcription
Oligonucleotide first binds to mRNA- short stretch of nucleotides contain lots of Ts which are complementary to polyA tail on sense strand
in reaxn Ts will bind to As on sense strand
Serve as site where reverse transcriptase can bind to end of Ts and synthesise copy DNA in 3’ to 5’ direction
Synthesises cDNA in 3’->5’ direction
produces anti-sense cDNA
cDNA amplified by PCR to produce ds DNA
uses specific primers
This is then separated where the sense strand is used to produce anti-sense RNA which is the probe
What is the point of the specific primers in the PCR amplification?
Amplifies just the single gene that interested in from a mixture of cDNAs as process of binding Ts to As not specific to any one gene
PCR primers used are specific for the one gene interested in
What else is done when designing the PCR primer?
Another RNA pol binding site added onto end of anti-sense strand DNA = T3
This is required to generate an RNA probe for in-situ hybridisation
What happens after ds cDNA made?
RNA pol separates 2 strands
uses sense strand as a template and produces antisense strand - complementary to sense strand mRNA present in tissue
This is the RNA probe
What modification is made to the nucleotides when the antisense RNA probe is being made?
Nucleotides used have DIG attached
How would in-situ hybridisation in a zebrafish work?
Have mRNA want to detect expression of in zebrafish
hybridise probe to mRNA
(probe = antisense RNA strand with DIG attached)
wash away anything not bound specifically
incubate with Ab bind to DIG
Ab have AP attached - add substrate
generates a purple colour within the embryo
What is trypan blue staining for?
Diazo dye used to colour dead tissues or cells selectively
Gives and idea of health of cells and percentage viability
What is the basis of trypan blue staining?
Based on negative charge - won’t interact with cell membranes unless they’re damaged
Living cells exclude the blue dye and appear bright under microscope
Dead cells take up dye and appear blue
Readily available, inexpensive and rapid
What is an MTT assay + what is its basis?
Colorimetric assay for assessing metabolic activity
NADPH dependent cellular oxido-reductase enzymes may reflect the number of viable cells present
Enzymes can reduce MTT to form purple, insoluble formazan crystals
Increased purple colouring = increased number of viable cells
Used in 96 well plates
Describe the process of bacterial transformation
Where bacteria take up a foreign genetic material (DNA plasmids) from the environment
These cells are known as competent cells
Plasmid vector with PCR DNA inserted is inserted into competent bacteria through electroporation or chemical transfer
Chem transfer = lipofection
or incubate with CaPO4 - has DNA associated and is insoluble in water, will precipitate
This is then taken up by endocytosis by bacteria
How is DNA (from pCR) inserted into a plasmid?
Through TA cloning
During amplification using Taq polymerase, it adds on an A at the end of the PCR product (3’end)
Plasmid vectors have T overhangs (3’) where they have been cut to be linearised
Are complementary to each other and A+T will form weak H-bonds
Hold structures in place, allowing DNA ligase to form phosphodiester bonds between them (ligating the DNA +vector together)
It also prevents the recircularisation of empty vector as T on one strand is not complementary to T on another strand
How are colonies selected for the inserted DNA?
Colonies are grown on selective media - ampicillin
to select bacteria have taken up the plasmid
If the DNA has been successfully inserted in the plasmid, it will destroy the lac-Z gene
Beta galactocidase enzyme won’t be produced by these bacteria and so when substrate X-gal added will not convert into blue product
Successfully transformed colonies = white colonies
Blue ones are unsuccessful
Activity of lacZ gene indicates whether DNA insert incorporated into plasmid or not
Prevents unnecessary screening of colonies - can just pick those that indicate have incorporated desired gene
How can desired DNA be extracted from bacterial cells?
Using a miniprep protocol + spin column
Put spin column in ependorf tube + spin in centrifuge
Wash
DNA will stick to resin on tube
everything else washed away + elute clean DNA from column itself
What is the purpose of bacterial transformation?
To produce multiple copies of the DNA
Don’t need to do PCR again, just go to stocks of bacteria bacteria
What are the principles of a Bradford assay?
Colorimetric assay based on absorbance shift of dye Coomassie Brilliant blue
Protein in assay = blue
No protein in assay = brown
Because in acidic conditions dye is converted from red to blue and will bind to the protein being assayed
What are the advantages of using zebrafish in research?
Genome similarities with humans
Embryos develop outside the mother’s body - easier to study early development
Grow at fast rate
Cheaper than mice
Embryos are transparent - can see development of internal structures
Hundreds of offspring at weekly intervals
Advantages of mice in research?
Similar to human genome
Easy to maintain - small so easy to house
Have short life cycle
Cheap
Relatively easy to manipulate genome - add/remove genes, to better understand role in body
Help study complex biological systems - immune system, nervous system
What is brightfield microscopy?
Sample attenuates light passing through it
Field outside the sample bright
Dye required in sample to attenuate light through it
+ve/-ve brightfield microscopy?
+ve - useful for things can stain + staining sections
-ve - can’t use for things can’t stain eg. live cell need something else
How does reverse transcription occur?
First strand cDNA synthesis
Requires mRNA, RT enzyme, primer + dNTPs
RNA incubated with primer at 70 degrees to denature RNA secondary structure
Then chilled on ice to allow primer to anneal onto RNA
RT, dNTP + buffer added to reaction
Then temperature changed to 37 degrees or higher to allow transcription to occur – 1hr
Then heated again above 70 degrees to denature enzyme = cDNA
How is the specificity increased?
Can generate gene specific primers - only make cDNA for specific target
yield is lower because targeting 1 transcript
How does PCR occur?
Heat up to 95 degrees and denature DNA – then do annealing stage by bringing temp down to 55-60 degrees, allows primers to bind to DNA
Heat again to 72 degrees to allow the polymerase to add the nucleotides starting from the primer + extend the molecule
This process is repeated multiple times to amplify the target DNA
Copies of target DNA will increase with each cycle, as will copies of variable length DNA fragments (but are a much lower proportion)
What must happen after a PCR reaction?
DNA must be purified - using gel electrophoresis
Separates mixed DNA fragments to estimate size or isolate fragments
And do DNA clean up protocol + elute DNA (spin column)
Give a brief outline of the steps necessary to generate double-stranded cDNA for a single gene, starting from a piece of tissue that is known to express the gene of interest.
Extract the mRNA from the tissue – using spin column and miniprep test kit
Then produce cDNA by putting the mRNA with random primers, RT enzyme, dNTPs and carry out reverse transcriptions – first strand cDNA synthesis
What is Western blotting used for?
To detect specific proteins in a sample of tissue extract
Identify specific proteins from a mixture of proteins extracted from cells
What happens in Western blotting?
Sample undergoes protein denaturation, followed by gel electrophoresis (to separate by size), visualisation (actual western blot) where mark target protein using primary and secondary antibody to visualise
Describe gel electrophoresis used in Western blotting
Electrophoresis is done using 2 gels –
Stacking gel pH6.8 (more acidic), separates protein poorly but allows form thin, sharply defined bands
Separating gel, pH8.8, pores are narrower, so protein is separated by their size more in this – smaller proteins travel more easily + rapidly than larger proteins
Proteins will travel towards positive electrode when voltage is applied
What does denaturation fo the protein allow?
Denaturing the high structure ensures that negative charge of amino acids is not neutralised, enabling the protein to move in an electric field – applied during electro-transfer
What happens after the gel has run?
After separation proteins are transferred to membrane through electric field oriented perpendicular to surface of gel
Semi-dry blotting
Transfer protein from gel onto PBDF membrane by using semi-dry blotter
Sandwich membrane with gel on top in between Filter paper
Clamp between 2 electrodes in blotter
Apply electrical current - protein within gel will migrate onto membrane
Fix protein onto membrane
How is the Western blot produced?
Introduce primary Ab to membrane, Ab recognises epitope to protein of interest
Then introduce secondary labelled Ab
2’ Ab labelled with Horseradish peroxidase
Incubate + wash off unbound Ab (PBS)
Add in substrate
Interaction between HRP + substrate allows light to be emitted so gives chemiluminescent signal
How can the light signal be visualised from the Western blot?
Visualise on X-ray film by exposure for few seconds/minutes
Should place x-ray film after reaxn occurs - get bands forming
What does HRP do?
In presence of hydrogen peroxide, converts luminol to intermediate dianion
Dianion emits light on return to ground state
For how long does light emission continue after exposure to substrate?
Maximal immediately after exposure
Continues 0.5-1hr after
What are some limitations of Western blotting/semi-dry blot?
Data produced is semi-quantitative – relative comparison of protein levels but not an absolute measure of quantity
Variations in loading + transfer rates between samples in separate lanes which are different between different blots
Need to be standardised before precise comparison can be made
Signal generated is not linear across concentrations in a range of samples - can’t be used to model the concentration
Why should they be transferred onto a membrane?
This enables incubation with different Ab allows pick up specific proteins within the extract
As up until that point there are a mix of proteins within the extract - helps quantitate specific levels of proteins within the extract
How do Bradford assays quantitate a reaxn?
Put the plate with the bradford assay in a plate reader + read at 595nm
Allows production of standard curve
From it can calculate amount of protein within a sample
How is the position of the ion in the gel determined in SDS-polyacrilamide gel electrophoresis?
Discontinuous gel system - where separating gel has different pH to stacking gel
Running buffer has glycine which has Hydrogen side chain
Position of ion dictated by pH hydrogen is running in
Stacking gel allows protein stack at fine line at the interface
Once protein enters separating gel - can be separated according to molecular weight
What components of the sample buffer help with loading?
Bromophenol blue Glycerol SDS Beta mercaptoethanol \+ sample buffer must be at right volume
What is the function of bromophenol blue?
Allows visualisation of sample, if only used this sample would float out of the wells
What is the function of beta mercaptoethanol?
Redcuing agent + reduces disulfide bonds in proteins - important to load samples onto system + allow it to migrate
What is the function of glycerol?
Adds density, so sample don’t float out of wells
What is the function of SDS?
Anionic detergent - breaks down non-covalent bonds
(as protein structure has lots of bonds - H bonds, ionic bonds)
Binds to amino acid side chains, giving protein net negative charge
important to separate proteins in the system so it can migrate to the positive anode
How to know when to stop running the gel?
Before loading, load a rainbow marker - mix of labelled proteins in different colours each corresponding to a different weight
Will know when to stop because know how big the molecules are
What can stained gels be use for?
Can stain all the protein in the extract - shows if sample equally loaded
does not identify specific proteins + difficult to quantitate
Equal loading of protein is required to be able to compare the wells
What are some positives of a semidry blot?
Semiquantitative - use software to quantitate bands gives an indication of whether there is an increase in the level of the protein or alteration in phosphorylation state
Can identify specific proteins w/extract
Can re-probe the blot - re-incubate membrane with another Ab to house keeping gene
eg. tubulin/gap-DH to show if have equal protein as they are proteins that shouldn’t be changing very much
What are the COSH regulations and how do they maintain safety?
Control of substances hazardous to health 2002 - COSH
Need to fill in the forms
Allow control of substances hazardous to health and prevent or reduce exposure to substance by:
Finding out what hazards are
Risk assessment - deciding how to prevent harm
Providing control measures to reduce harm to health and ensuring they are used
Providing information, instruction and training for employees
Planning for emergencies
What are adherent cells?
They are grown in a monolayer
and can be released from dish/flask enzymatically/mechanically
Growth is limited by surface area
What are non-adherent/suspension cells?
Grow freely in liquid as single cells/free floating clumps
Can grow to high density in very small volume
What is density with regard to cell culture?
Measure of proliferation
When gets too dense for flask, must be passaged (sub-cultured)
What is confluency?
Combined with density - estimate or counted percentage of dish/flask covered with cells
100% entirely covered
10% confluency = 10% surface covered
Sub confluent level = 30-40% allows space for doubling up
What is passaging and what is the passage number?
Passaging/subculture is lifting cells, diluting the volume and removing cells (enzymatically) .˙. maintaining the stock of growing cells
Passage number = number of times cell culture has been subcultured - some cell lines only grow for finite number passages
Describe enzymatic removal of cells
Use trypsin-EDTA
Trypsin cleaves peptide bonds in fibronectin of extracellular matrix (would attach cells to surface/plastic)
EDTA chelates Ca in media
Ca would normally inhibit trypsin
What is the danger of using trypsin for too long?
Can reduce cell viability
Can alter phosphorylation state some proteins found at the cell surface
(if looking at phosphorylation state, should transfer cells into centrifuge tube and produce cell pellet - can lyse pellet)
How are cells mechanically removed?
Using a cell scraper
What happens once the cells are detached?
Quantitated using a haemocytometer to determine cell density
Count cells in outermost 4 squares + use formula to work out how many cells/ml
Once know cell density/number can seed cells for experiment
If want to compare experiments, should seed cells at same number/density each time
What can cell number be used for?
To determine how well cells grow - enables plot growth curve
How are proteins isolated from a cell and where can they be extracted from?
Use detergents to isolate protein
Different strength of detergent isolate from different regions eg. nucleus/cytoplasm
Whole cell extract - lyse cell + isolate cell extract
Cytoplasmic cell extract
Nuclear cell extracts
What are the different types drug screens and what type of research are they an example of?
Examples of exploratory research
Phenotypic drug screens:
morphological - identifies compounds that lead to a particular morphological effect
Therapeutic - identify compounds that ameliorate a disease phenotype
use model organisms where human disease features are reproduced, treat with compounds and see which are reverting disease phenotype - can be further developed into therapeutic treatment
pathway directed - know that in a disease particular signalling pathway is affected, want to find compounds would target the pathway
identify compounds that interfere/modify the activity of specific molecules/signalling pathways
give data or tools that can use for hypothesis driven research
What is exploratory research?
Increase knowledge which can lead to an experiment
Not driven by initial hypothesis
What type of research is used in labs?
A mix of hypothesis and exploratory research
What is hypothesis driven research?
Observe a phenomenon/identify problem
Generate hypothesis - answer to question, idea as to what happens
Design experiments to test hypothesis
Allows acceptance or rejection hypothesis
How is protein synthesis regulated in the body?
Regulated at 2 levels
Transcript
Global regulation
Describe regulation at the transcript level
Message/transcript - as eukaryotic mRNA has innate characteristics which influence how easily transcript is translated by ribosome
eg. 5’UTR has many secondary structures which can inhibit/affect the efficiency that the ribosome scans through 5’ UTR before reaching the start codon
Describe global regulation
Affects whole process within the cell
eg. tissue culture being grown in an artificial environment cells are extremely sensitive to that environment
Allow them to be overcrowded .˙. confluency is too high, allows them to be stressed
Will begin to shut down protein synthesis
What are some factors that cause cell stress?
Withdrawal of nutrients Overcrowding Temp shock Starve by removing amino acids from tissue culture media Hypoxia Viral infection
What happens as a result of cell stress?
Shut down of protein synthesis
If stress prolonged, cell will activate other pathways eg. apoptosis
Describe translation
Very fast process - to supply protein in cell
Can link 400 aa in 20 seconds
Slow enough to avoid too many errors
Error rate = 1 in 10^4 incorrect amino acids
What is peptidyl transferase activity associated with?
Associated with 60s subunit
links aa together during elongation
When is the potential to initiate protein synthesis the greatest?
When the 2 ribosomal subunits are dissociated
As 40s needs to be trapped by eukaryotic initiation factor will allow it to enter process of initiation
What is the principle behind confocal microscopy?
Laser light is focused onto defined spot at a specific depth within the sample
This causes emission of fluorescent light at exactly this point
A pinhole inside the optical pathway cuts off out-of-focus signals allowing only fluorescence signals from the illuminated spot to enter the light detector
Scanned in a raster pattern, producing imaged in a single optical plane
How can 3D objects be visualised using confocal microscopy?
Scan multiple optic planes + stack using a suitable microscopy deconvolution software
What else is possible with confocal microscopy?
Analysis of multicolour immunofluorescence stainings using state-of-art confocal microscope that include several lasers + emission/excitation filters
What is confocal microscopy used to resolve?
Detailed structure of specific objects within the cell
Components of living or fixed cells can be specifically labelled using immunofluorescence and then visualised in high resolution
What is a positive of confocal microscopy?
Produces sharp images of the exact plane of focus, w/o any disturbing light from the background or other regions of the specimen
Thicker objects can be visualised
3D structures can be visualised
What is a limitation of confocal microscopy?
Sample penetration depth is limited - thicker specimens should be imaged using two-photon microscopy/LFSM
What is widefield fluorescent microscopy used for?
For the detection of specific structures, molecules, or proteins within a cell
Almost any component of living/fixed cells can be stained + specifically imaged
What type of assays are widely analysed using widefield fluorescence microscopy?
Immunofluorescence assays
What else can be done with widefield fluorescence microscopy?
Can visualise several structures in parallel, which then appear as different pseudocolours in the obtained image
What is the principle of widefield fluorescence microscopy?
Based off of light microscopy
Fluorophore coupled Ab will bind to the specific structures within the cell
Specimen illuminated at an excitation wavelength and viewed through a filter only allowing the emitted wavelength through
Because when illuminated the fluorophore will absorb the light at a specific wavelength and then emitted at higher wavelength
The background will be dark and structures with bound fluorophore will emit light, indicating presence of structure of interest
What are some limitations to widefield fluorescent microscopy?
Whole specimen exposed to light - fluorecence signals from all focal planes are detected, leads to lower contrast in thick samples like spheroids + tissue
When is widefield microscopy best applied?
Thin specimens with low background autofluorescence
eg. adherent cells
What colour is phalloidin and DAPI?
Phalloidin - green, F-actin
DAPI - blue, stains nuclear DNA
How is cross hybridisation decreased in in-situ hybridisation?
Using a high hybridisation temperature + formamide in HM buffer ensures high stringency of hybridisation
How is non-specific hybridisation of the probe avoided?
Using high stringency washes - 0.2x SSC at 70degrees
For single locus/large probes - temperature should be higher (65 degrees) + stringency high (SSC below 0.5)
Washing for longer times can wash away more excess probe - too long may risk washing off too much hybridised probe
What does Accutase do?
Similar to trypsin EDTA - allows for detachment of cells from surfaces
Auto-inhibits at 37 degrees without need for neutralising solution
How are proteins extracted for western blotting?
Remove the cells needed by mechanically scraping (+use the media as it may contain cells)
Centrifuge at 4 degrees for 10 mins - discard supernatant
Re-suspend pellet in 1ml of PBS + transfer to labelled eppendorf tube
Spin down at 11,000 rm for 10 seconds - discard the supernatant
Re-suspend in 35ul of lysis buffer, pipette up+down to break up pellet
Vortex (5sec) + incubate on ice
Then sonicate 3 times to break open cells (using sound energy)
Spin in chill spinner at 14,000g for 10 minutes at 4 degrees
Transfer supernatant into new labelled eppendorf tube
What is the role of BSA in Bradford assay?
BSA is used as a standard for the Bradford assay, and a calibration curve based on the mass concentration of BSA used to determine unknown concentrations of a protein.
What is a biotinylated marker?
Biotinylated protein ladder detection pack is designed to detect the molecular weight ladders on Western blots when using the horseradish peroxidase (HRP) based Western detection system.
The pack contains Biotinylated Protein Ladder and Anti-biotin, HRP-linked Antibody
What is the purpose of TEMED and Ammonium Persulphate in Western blotting?
Both are used to catalyse acrylamide gel polymerisation when preparing gels for gel electrophoresis
How are proteins stacked and then separated?
Stacking gel is usually formulated at pH 6.7–6.8.
At this pH, ionized chloride ions migrate rapidly, raising the pH behind them and creating a voltage gradient with a zone of low conductivity
This causes glycine (from the running buffer) to ionize and migrate behind the chloride front. Most peptides in the sample, which have a negative charge due to the bound SDS, migrate between the chloride and glycine, forming a narrow band and thus becoming “stacked”.
Once the stack reaches the resolving gel, which is at a higher pH (typically pH 8.7–8.8), the increased ionization of the glycine causes it to accelerate and overtake the peptides. Smaller pore size of the resolving gel starts to have a sieving effect, resulting in the separation of peptides by size.
Most western blotting protocols use a Tris buffer of low ionic strength for protein transfer. The transfer time depends on the type of blotting apparatus and the peptide size range of interest.
How are proteins stacked and then separated?
Know the purpose of Tris- HCL
Stacking gel is usually formulated at pH 6.7–6.8.
At this pH, ionized chloride ions migrate rapidly, raising the pH behind them and creating a voltage gradient with a zone of low conductivity
This causes glycine (from the running buffer) to ionize and migrate behind the chloride front. Most peptides in the sample, which have a negative charge due to the bound SDS, migrate between the chloride and glycine, forming a narrow band and thus becoming “stacked”.
Once the stack reaches the resolving gel, which is at a higher pH (typically pH 8.7–8.8), the increased ionization of the glycine causes it to accelerate and overtake the peptides. Smaller pore size of the resolving gel starts to have a sieving effect, resulting in the separation of peptides by size.
Most western blotting protocols use a Tris buffer of low ionic strength for protein transfer. The transfer time depends on the type of blotting apparatus and the peptide size range of interest.
What is LiCl used for in riboprobe preparation?
Precipitation of the RNA probe
What is PFA (paraformaldehyde) in in-situ hybridisation?
Used to fix cells
What is methanol used for in in-situ hybridisation?
Dehydrate the cells (to allow storage?)
What is proteinase-K used for in in-situ hybridisation?
Embryos are prepared for WISH by treatment to ensure that the probe will reach even the least accessible parts of the embryo.
This is most critical to ensure accessibility in zebrafish embryos that are 24hpf or older
What is Hyb+/Hyb-used for in in-situ hybridisation?
Hyb+: 5XSSC, 50%formamide, 0.1%triton, 5mg/ml torula RNA, 50ug/ml heparin
Hyb-: 5XSSC, 50%formamide,0.1%triton (at 68°C)
What is SSC used for in in-situ hybridisation?
Saline-sodium citrate (SSC) buffer is used as a hybridization buffer, to control stringency for washing steps in protocols
What is triton used for in the preparation of the staining buffer in in-situ hybridisation?
Permeabilises the cells/tissue to allow them to stain
Role of glycerol in in-situ hybridisation
For storage
What is Ethidium bromide used for in DNA gel-electrophoresis?
Binds to DNA + fluoresces when exposed to UV rays
What is IPTG used for?
It functions as an inducer of galactosidase activity by binding to and inhibiting the repressor. It is utilised for the induction of expression from the lac promoter and derivates.
eg. Blue/white colony selection is often used to increase the likelihood of recovering desired DNA constructs following ligation into a plasmid vector. What factor is responsible for the induction of beta-galactosidase protein in this process?
A = IPTG
What is X-gal?
X-Gal is used to detect the insertion of foreign DNA into the lacZ region of a plasmid DNA.
Converted into blue product if lac-Z gene intact and B-galactocidase is made to convert it
