Protein-protein interaction study: Binding analysis

Protein-protein interaction study: Binding analysis


Welcome to the MOOC NPTEL course on intractomics
To completely understand the function of a protein it is important to look beyond its
expression pattern and identify its potential interaction partners and determine their interaction
dynamics To study the interaction between two binding partners in SPR experiment one
partner is attached to the surface and other is passed over the surface in a continuous
flow of sample solution The interaction of ligand and analyte is measured
by the SPR instrument as a change in the refractive index over time and response observed is directly
proportional to the change in mass concentration close to the surface In the previous lecture
we completed immobilization procedure of anti-beta two microglobulin antibody on the surface
of a CM5 chip we will now move forward to perform the binding analysis of anti-beta
two microglobulin antibody with beta two microglobulin protein So let us have the experimental session now
We will go into the second session now and we will use our immobilized chip for a binding
experiment So here we will first prepare a template for the binding experiment A binding
experiment actually deals with screening or a single concentration screening of different
compounds on the immobilized ligand and look for its ability to interact with the target
that is immobilized on the surface Before we go ahead with the binding experiment
let us understand some important considerations during sample injection the analyte an injected
over the surface with a constant flow and concentration Analyte in the sample binds
to the immobilized ligand on the surface the mass on the surface changes and the response
is recorded After sample injection buffer flows over the surface to allow monitoring
of analyte dissociation from the ligand Regeneration as already discussed by Dr Srinivas
is the process of removing bound analyte from the ligand on the sensor chip surface after
analysis of a sample Efficient regeneration which means removing bound analyte without
affecting the ligand activity is crucial to a successful assay If the regeneration is
incomplete or the binding activity of the surface is reduced the performance of the
assay is impaired The choice of conditions for regeneration
is dictated by the stability and nature of the ligand and analyte In today’s binding
experimental setup we will be using HEPES-EP plus as the running buffer we will preparing
three different concentrations of beta two microglobulin protein which are 8.5 nanomolar
of 42.5 nanomolar and 85 nanomolar for evaluating it is binding with the antibody having 8.5
nanomolar concentration in duplicate These three concentrations will be referred to as
low medium and high Contact time between the sample and the sensor
surface should be sufficient to give confidently measurable response levels without compromising
screening throughput Contact time of 22 minutes are usually sufficient for a binding experiment
Here we will provide a contact time of 60 second at the flow rate of 10 microliter per
minute with the dissociation time of 60 seconds And ideal regeneration condition is the one
their analyte response of the same concentration is constant after repeated injections Today we will be using 10 milimolar glycine
pH 2.5 for regeneration of the surface; we will now proceed with our binding experiment
protocol Well before making the template we will open the file wizard again binding analysis
new identify the flow path as 2 minus 1 as we have done our immobilization on 2 minus
1 chip will be CM5 that is docked already we will not have ligand capture sample and
regeneration We will go to the next tab here we are not
using any conditional cycle we will start with the startup cycle Startup cycles are
cycles of buffer uhh used for equilibrating the system So here basically buffer is used
as analyte So we can type as HEPES-EP plus buffer and from the pull down menu we will
select 3 cycle Generally for binding experiments 3 cycles are selected going to the next tab
for setting up of binding we need to specify the contact time as 60 seconds default flow
rate of 10 microliter per minutes Dissociation time of any minute or any second by default
we can consider 60 seconds The regeneration solution we would prefer
here would be 10 milimolar glycine pH 2.5 with the default contact time of 30 seconds
flow rate of 30 microliter per Minuit and with no stabilization time We go to the next
tab; here we need to fill in the name of all single concentration compounds So here we
would select our analyte as beta two M just that we have one analyte we will take it in
three different concentrations So we will name as low beta two M medium and beta two
M high So low indicates lower concentration medium
medium concentration high concentration and we will go to the next tab we will collect
prime before run and normalize is not required here because the chip is already immobilized
and we will go with the default temperatures and we will go to the next tab here we will
not select a micro-titer plate and this is our rack positions for a binding experiment
Here we have at the C1 position HEPES-EP plus buffer for three different startups We have
three concentrations of analyte; high medium and low and we have the regeneration solution
here which is 10 milimolar glycine pH 2.5 and we will prepare our solutions and start
the binding experiment We will now work on the reagent require for
the binding analysis of anti-beta two microglobulin with beta two microglobulin proteins We will
be using HEPES-EP plus as the running buffer which will also be used for the initial startup
cycles We will tell you the stock solution of protein that is 100 microgram per ml in
the running buffer HEPES-EP plus to prepare 100 microliter of three different concentrations
that is 85 nanomolar 42.5 nanomolar and 8.5 nanomolar which are referred to as high medium
and low concentrations in the experiment We will also include one zero nanomolar concentration
in the experiment which will be nothing but the running buffer For the regeneration of
the surface we have prepared glycine HCl pH 2.5 as the regeneration solution we have transferred
all the solution in the specialized tube starting from the startup beta two M concentrations
starting from 85 nanomolar 42.5 nanomolar 8.5 nanomolar and zero nanomolar The regeneration solution is placed in this
glass vial We will now insert these tubes into the appropriate rack and then into this
system to start with the binding analysis of anti-beta two microglobulin with beta two
microglobulin protein We will eject the rack now to insert new vials eject
rack and take the plate out of the sample rack
and we will create with the binding vials. So the vials positions filled with different
samples as you can see on this screen the startup is here So beta two M medium beta
two M low beta two M high are put at their respective positions and vial for regeneration
of 10 milimolar glycine pH 2.5 is here Now we close the rack and will be inserted in
the sample compartment by ejecting the rack compartment inserting the plate next tab again
we need to do all these checks check the prime The estimated run time of 38 minutes and we
have sufficient amount of buffer and we will now start the experiment; we will save this
template as binding save Now we will save the result vial again as binding And now the
experiment has started shows running binding analysis with an estimated time of 38 minutes
System is priming now and once we finish off the binding experiment we will take a look
at the data Before we analyse the binding data from the
experiment let us look at a typical sensorgram for binding between a ligand and an analyte
A sensorgram as shown here is a plot of response against time showing the progress of interaction
This curve is displayed on the system during the course of experiment we observe the base
line followed by the injection of analyte which leads to increase in the binding response
during the association phase Just after this top of the sample injection
we observe report point which records the response on a sensorgram at a specific time
averaged over a short time window This is followed by dissociation phase regeneration
and then back to baseline We will now proceed to analyse the data obtained
from binding of anti-beta two microglobulin with beta two microglobulin protein after
finish of our binding experiment by double click on the file the file is open now we
will see here from our binding experiment it shows all sensorgram here So here the green
ones are our startup So we have setup 3 or 5 different startups and the red ones are
our actual data from beta two microglobulin What we will do is we will highlight only
our sampled data And now you see the data for beta two M We
go on to sensorgram adjustment to report point on the vial adjustment baseline and save ok
Now our data is baseline to zero if we want we can as well go on to color sample and we
will see the different samples in different color with the ligand on one side of the screen
Here we have low medium and high concentrations of beta two microglobulin injected over anti-beta
two M; we could subtract or delete the regeneration area cut Now we can see our different concentrations
of beta two M with one of them in duplicate So there is definitely binding of the beta
two M to anti-beta two M antibody in a dose dependent manner The data can also be shown
in the form of a bar chart with all our red startup runs and green as our sample runs
Here we will highlight the sample runs only and see cycle 78 and 9 are our low medium
and high concentrated data which are dose dependent binding Going to another tab we can see the relative
responses of each molecule from the binding at 4 for the duplicates and medium at 29 and
high at 52 With this we will conclude our binding session and we prepare now for a kinetics
experiment Protein interactions are identified using
a wide array of applications however what is also required is a understanding of the
extent to which these interactions actually occur Therefore performing protein-protein
interactions studies and calculating their kinetics values becomes very crucial Let us
continue our discussion and SPR experiment for the kinetics analysis in next lecture
Thank you