Lecture 15 - Protein Interactions I

Question 1

functional product of genes:

Answer 1

protein

Question 2

proteins interaction allows them to:

Answer 2

• create surfaces for cellular function
• create, alter or break down molecules (protein, DNA, RNA or metabolites)

Question 3

antibodies:

Answer 3

multi-protein molecules made by immune system to remove foreign molecules called antigens

Question 4

the dimer of antibody heavy chains end with:

Answer 4

an antigen specific binding site which recognises specific molecules

Question 5

new use of antibodies:

Answer 5

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

Question 6

immunoprecipitation:

Answer 6

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

Question 7

what is the name of the protein we are targeting to pull down in immunoprecipitation?

Answer 7

the target pull-down protein is sometimes called ‘the bait’

Question 8

how do we bind antibodies to the ‘beads’?

Answer 8

we can connect antibodies to the beads using covalent bonds

Question 9

what are the two forms of bead attachment?

Answer 9

(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

Question 10

once the protein has bound to the beads via the antibody…

Answer 10

… we can then separate the complex through adding water - this elutes the antibody and protein from the beads

Question 11

in recent years what has really helped the process of immunoprecipitation?

Answer 11

the use of magnetic beads which allow for efficient clearance

Question 12

how do magnetic beads function?

Answer 12

the magnet attractors the beads to the side of the tube and hold them there allowing the rest of the sample to be removed

Question 13

how do you separate the antibody and target protein?

Answer 13

you can heat the solution to dissociate the antibodies to the bound protein

Question 14

co-immunoprecipitation:

Answer 14

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

Question 15

transient vs stable protein interactions:

Answer 15

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’

Question 16

how can we overcome the difficulties that come with unstable, transient proteins during immunoprecipitation?

Answer 16

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

Question 17

what is another way of increasing the pull-down rate with transient proteins?

Answer 17

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

Question 18

what can cross linking proteins also be used for?

Answer 18

cross linking can also be used to help you in determining secondary protein structure

Question 19

SDS-PAGE:

Answer 19

• 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

Question 20

western blots

Answer 20

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

Question 21

secondary antibodies:

Answer 21

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

Question 22

how to interpret an IP Western:

Answer 22

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

Question 23

what must we know in order to carry out a western blot and how can we do this?

Answer 23

you must know what protein you are examining, to identify this protein you can use mass spectrometry (which can identify multiple at once)

Question 24

chromatin immunoprecipitation:

Answer 24

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

Question 25

what can chromatin immunoprecipitation be used for?

Answer 25

tool in gaining information regarding transcription factors, chromatin binding and histone modifications

Question 26

cross-linking immunoprecipitation (CLIP):

Answer 26

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

Question 27

chromatin & cross-linking immunoprecipitation and disease;

Answer 27

can quantify easily whether a mutation in a protein that binds to nucleic acid changes what it binds to

Question 28

increase chances of pulling through a protein and its interaction partners:

Answer 28

through using crosslinking or overexpression

Question 29

RNAi

Answer 29

knock out not knockdown = incomplete protein degradation, transient, off target effects, fast and easy

Question 30

sanger

Answer 30

high accuracy

low thoroughput

Question 31

NGS

Answer 31

lower accuracy

very high thoroughput

Question 32

nanoplre

Answer 32

ultra long read lengths

higher bp error rate

Question 33

other protein interactions:

Answer 33

• screen for other interacting proteins using mass spec
• DNA sequences in genome (ChIP-seq)
• RNA interactions (CLIP + ChIP-sec)

Question 34

immunofluorescence and colocalisation:

Answer 34

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