Lecture 15 - Protein Interactions I Flashcards
functional product of genes:
protein
proteins interaction allows them to:
- create surfaces for cellular function
- create, alter or break down molecules (protein, DNA, RNA or metabolites)
antibodies:
multi-protein molecules made by immune system to remove foreign molecules called antigens
the dimer of antibody heavy chains end with:
an antigen specific binding site which recognises specific molecules
new use of antibodies:
just as we can use antibodies for immunofluorescence to see expression and localisation of proteins, we can use them for immunoprecipitation to find out what a protein is interacting with
immunoprecipitation:
uses antibodies to selectively bind to our protein of interest and then to precipitate or pull them down this means we are able to pull out of a sample one specific protein
what is the name of the protein we are targeting to pull down in immunoprecipitation?
the target pull-down protein is sometimes called ‘the bait’
how do we bind antibodies to the ‘beads’?
we can connect antibodies to the beads using covalent bonds
what are the two forms of bead attachment?
(1) you can either bind the antibodies to the beads first then allow them to react with the antigen
or
(2) let the antibody bond to the protein first and then allow the antibody to bind to the bead
once the protein has bound to the beads via the antibody…
… we can then separate the complex through adding water - this elutes the antibody and protein from the beads
in recent years what has really helped the process of immunoprecipitation?
the use of magnetic beads which allow for efficient clearance
how do magnetic beads function?
the magnet attractors the beads to the side of the tube and hold them there allowing the rest of the sample to be removed
how do you separate the antibody and target protein?
you can heat the solution to dissociate the antibodies to the bound protein
co-immunoprecipitation:
when we pull down our protein of interest, the protein is probably active and may be interacting with its target proteins, if the pull down doesn’t interrupt that interaction, when we take our antigen of interest we also take anything that is bound to it - this means our elution will not only have proteins of interest but also any proteins that are attached to it
transient vs stable protein interactions:
some proteins are required to stay bound to each other for a complex to be active (e.g: molecular motors)
other proteins only need to be bound for a short period of time to be functional (e.g: kinases)
strong stable interactions result in easy ‘pull-down’ whilst weak transient interactions result in difficult ‘pull-down’
how can we overcome the difficulties that come with unstable, transient proteins during immunoprecipitation?
one way to increase picking up a transient interaction is to overexpress interacting proteins
this can be done through cloning the gene of interest, inserting into cells and allowing the protein to be produced and then undertake an IP
more protein = more chance of pick up reaction
what is another way of increasing the pull-down rate with transient proteins?
the other main way to improve pick-up is in the form of cross-links: this normally involves using UV or chemicals like paraformaldehyde
cross linking is the addition of bonds that will link molecules together more tightly - this results in the interaction being stabilised so it does not dissociate
what can cross linking proteins also be used for?
cross linking can also be used to help you in determining secondary protein structure
SDS-PAGE:
- proteins being separated using a technique called sodium dodecyl sulfate polyacrylmide gel electrophoresis
- the SDS detergent and heat denatures the proteins which unfolds them and gives them a negative charge
- these proteins then migrate through a polyacrylamide get matrix when an electrical current is applied towards the positively charged end
- the larger the proteins the slower it moves through the gel matrix, thus separating proteins by size
western blots
present antibodies against proteins of interest on our gel to specifically see if the proteins are present in sample
can do in the gel but not very efficient as we need a lot of antibodies which are very expensive, so we actually take the proteins in the gel and put on a membrane in order to reduce the amount of antibody required massively
first you add a primary antibody and then a secondary antibody in order to detect binding
you should only get one band on your gel
secondary antibodies:
secondary antibodies are antibodies which bind to other (primary) antibodies - they can be tagged with fluorophores so that we can detect the binding on our gel membrane
how to interpret an IP Western:
essentially the interpretation of different protein bands
IP band:where the protein of interest has been pulled down or removed from the input sample along with any proteins bound to it - this is where we see whether the proteins interact with each other
input band: this is the origional sample with all the protein - this allows us to make sire that the proteins we are interested in are expressed in the sample
unbound: sample we remove after we have done the protein pull down with antibodies and beads - this can give us indication of both efficiency & the level of interaction
IgG band: this is just the base antibody - this is to make sure that the bands that we are seeing are not just antibody we used for the pu;; down
what must we know in order to carry out a western blot and how can we do this?
you must know what protein you are examining, to identify this protein you can use mass spectrometry (which can identify multiple at once)
chromatin immunoprecipitation:
looking at the interaction between proteins and nucleic acids
crosslinks the DNA and proteins together, specifically bind our protein of interest, denature to remove the antibody and sequence the DNA
what can chromatin immunoprecipitation be used for?
tool in gaining information regarding transcription factors, chromatin binding and histone modifications
cross-linking immunoprecipitation (CLIP):
using cross linking can allow you to examine RNA through cross-linking, pull-down and RNA sequencing
useful for looking at what RNA-binding proteins are binding to
chromatin & cross-linking immunoprecipitation and disease;
can quantify easily whether a mutation in a protein that binds to nucleic acid changes what it binds to
increase chances of pulling through a protein and its interaction partners:
through using crosslinking or overexpression
RNAi
knock out not knockdown = incomplete protein degradation, transient, off target effects, fast and easy
sanger
high accuracy
low thoroughput
NGS
lower accuracy
very high thoroughput
nanoplre
ultra long read lengths
higher bp error rate
other protein interactions:
- screen for other interacting proteins using mass spec
- DNA sequences in genome (ChIP-seq)
- RNA interactions (CLIP + ChIP-sec)
immunofluorescence and colocalisation:
primary antibody for protein of interest and then you add another secondary antibody that is specific to the primary antibody that has a glowing fluorophore attached to it
