WEEK 5 Flashcards
Recombinant DNA technologies
we can manipulate the protein we’re interested in inside the cell
- we can force the cell to make extra copies of the protein
- we can remove the protein that the cell naturally makes to assess its function
- we can make the cell create mutant versions of the protein to work out what part of the protein is important for its function
- we can make tagged versions of the protein to find it among other proteins in the cell
Plasmid vector
circular piece of DNA containing:
1) the gene for the protein we’re interested in, the exact sequence without introns or up/downstream sequences - this means you can only express one isoform for the protein
2) the promoter for that gene
3) an antibiotic resistance gene
4) a eukaryotic resistance marker
Polymerase chain reaction (PCR)
tool we use for copying or amplifying the gene in the original plasmid, so we can clone it into the desired plasmid
DNA denaturation
heating a sample to a high temperature breaks the hydrogen bonds holding the two strands of the template DNA together, so that the double helix splits open, exposing the bases on the inside
Primer annealing
the denatured DNA is cooled, which allows the primers to bind to the start and end of the DNA sequence we want to amplify
Primer extension
the mixture is then heated to the optimum temperature for the DNA polymerase to work on the exposed DNA strand. the polymerase slides along the strand and links bases together, synthesizing a new strand of DNA. This doubles the amount of DNA.
Amplifying the gene
we simply repeat the cycle of heating, cooling, and warming the DNA. with each cycle, we double the number of copies we had. the process is exponential, meaning we can very quickly amplify our target piece of DNA.
Ligation reaction
we use a DNA ligase to close the bonds. this connects the inserted DNA to the plasmid, completing it.
Bacteria transformation
we place the plasmid and the bacteria on an agar plate, heat them to 42 degrees then immediately cool them to 4 degrees in ice. the thermal shock causes the bacteria to take up the plasmid from their surroundings. bacteria transformation occurs when bacteria take up exogenous DNA.
SDS-PAGE
method that uses electrophoresis to separate proteins in a sample by molecular weight. it works by applying an electrical field across a gel made by a molecule called polyacrylamide.
steps:
1) break down the tissue sample to release the proteins. proteins are then denatured and given an electrical charge
2) the proteins are then placed in small wells at one end of the gel. when the electric field is switched on, the molecules are attracted to the end of the electric field that has the opposite charge. smaller molecular weight proteins migrate across quickly, and larger molecular weight proteins migrate slower.
3) the proteins are spread out and have been separated
SDS-PAGE: mechanical breakdown
homogenizing the cells in a homogenizer or sonicating them by blasting them with ultrasonic vibration. this tears the tissue apart and rips open the cell membranes, letting all the proteins out
- preserves structure
SDS-PAGE: chemical breakdown (lysis)
mixing the cells with a buffer that will break the membranes apart
- does not preserve structure
SDS molecule
acts as a denaturing agent.
- has a long hydrophobic tail and a negatively charged head. the tail binds to the protein and pulls apart its structure, thereby unfolding it. because of its negative charge, once bound to the protein, it renders it negative too.
SDS-PAGE: gel
the gel is made by cross-linking lots of acrylamide molecules together. it is composed of pores of many different sizes, which helps to separate the proteins as the larger molecules can’t fit through the smaller pores so easily.
- gel: acrylamide is mixed with an SDS-containing buffer. then, 2 catalysts are added to initiate a polymerisation reaction that makes the acrylamide molecules cross-link.
SDS-PAGE: resolving and stacking gel
- the stacking gel is made up of a lower pH and lower % of acrylamide than the resolving gel
- when proteins hit the join between the stacking and resolving gel, the change in pH and % of acrylamide slows them down. the proteins entering the resolving gel first are slowed down first, allowing the proteins entering after to catch up.
- if we didn’t use stacking gel, the proteins would all enter the resolving gel at different times, resulting in large and smeared bands.
- we can alter the % of acrylamide in the resolving gel depending on the size of protein we want to look at. a lower % means bigger pores means a better resolution of larger proteins. a higher % means smaller pores means a better resolution of smaller proteins.
Western blotting
- method we use to detect specific proteins from a mixture of proteins following an SDS-PAGE.
- we transfer the proteins from the gel onto a membrane which blots the proteins from the gel. we sandwich the gel and membrane between electrodes, apply current, and since the proteins are still negatively charged, they migrate from the gel onto the membrane (direction of the positive electrode).
- once they’re on the membrane, they’re immobilized thanks to nitrocellulose and PVDF in the membrane which have good protein retaining properties
- allows us to assess the amount of a specific protein in different samples
Western blotting: advantages
- quick, only taking about 1.5 days
- does not require a lot of specialist equipment
- works for a wide range of proteins, all you need is a good antibody
- can be quite sensitive and specific with good antibodies
- semi-quantitative: if you see a bigger signal, it means you have more protein. you can thus use it to look at changes in protein levels
Western blotting: disadvantages
- it is only as good as the antibody
- antibodies can be very expensive
- many antibodies do not bind specifically, making results confusing to interpret. in addition, an antibody may not work once a protein has been denatures. this is why you have to tag it with a short sequence, which antibodies bind well to.
- sometimes proteins become modified by the cell once they’ve been synthesized, making them hard to interpret
- does not work as well if your proteins are very large or very small: large proteins are slow and don’t transfer well to the membrane, requiring a low % gel which is hard to handle and doesn’t transfer well. small proteins run fast and need high % gels and can easily run off the end of the gel or through the membrane in the transfer
- you need a change of at least 10% in the levels of a protein for it to be detected
- semi-quantitative: you can only see approximate changes
