Protein immobilization for protein-protein interaction studies

Protein immobilization for protein-protein interaction studies

Welcome to MOOC NPTEL course on intractomics
today we will talk about protein-protein interactions study immobilization of the ligand on sensor
chip Surface Plasmon resonance has transformed the study of bio-molecular interactions by
delivering a platform that does not require the ligand or analyte to be labeled SPR measures
the interaction between a ligand which is immobilized on the sensor chip surface and
an analyte which is passed in the solution This measurement takes place in real time
label-free environment providing kinetic equilibrium and concentration data In today’s SPR immobilization experiment
the ligand anti beta 2 microglobulin will be covalently immobilized to the surface of
sensor chip using amine coupling chemistry The direct immobilization of the ligand is
known as direct coupled Another way of immobilizing a bio-molecule is by using capturing chemistry
In this case the ligand is not covalently immobilized to the sensor chip surface but
is captured through the electrostatic interactions The major steps involved in the immobilization
of anti beta 2 microglobulin antibody will involve activation preparing for amine coupling
immobilization and deactivation So let us have the lab experimental session now Let
us learn a little about the basics of immobilization and SPR assay In the molecular interaction
study using surface plasmon resonance we will immobilize one of the interacting molecule
that is anti-beta two microglobulin on the gold sensor chip surface while the protein
beta two microglobulin will be passed over that surface in solution Here the ligand refers
to the immobilized component and the interacting partner in the sample injected over the surface
is referred to as the analyte The three major steps in a biacore SPR assay
involved; immobilization the process by which ligand is attached to the sensor chip surface
interaction analysis where the analyte is injected over the sensor chip surface and
the interaction between the analyte and immobilized ligand is monitored And regeneration the process
of removing bound analyte from the ligand on the surface So this is a surface Plasmon resonance and
it is a biacore T200 So this machine has the various parts like the running buffer is connected
here and any of the biological buffers can be connected as a running buffer There could
be have any pH from low to high So the regular buffers used in a biacore experiment are HBS
EP-HBSN PBS for small molecule experiments Coming here we have a water reservoir The
water reservoir is useful for cleaning niddles and syringe and we have a waste reservoir
here and this waste reservoir collects all the waste The samples are actually sent in
this compartment chip is docked in this compartment So the experiment starts by picking up samples
from this compartment and transferring them here at the interaction side and then the
experiment is recorded on the screen Here some kind of indications are provided when
the machine is ready or the system is calibrating temperature when the new chip is docked and
the run is actually happening So we will now connect a new running buffer
here and prime the system before our immobilization experiment So here the new HBS-EP plus buffer
is connected and the new chip will be docked now So let us look at the chip now So this
is a new chip the chips are generally provided in these cassettes and then new CM5 chip will
look like this This chip will be inserted in chip docking area
from the control software we will eject to the sensor chip that is connected So now the old chip will be removed and we
will insert a new biacore chip CM5 here and the insertion or the way orientation of the
chip is shown on the chip in arrows and we will close the compartment door and
identify the chip from the chip type here and chip will be given a new id and sometimes
it is very essential to add the name for the chip and also the lot number
and save dock chip Now the chip is getting docked So we have
connected a new buffer we will prime the system Priming is the process of sending buffer through
the IRC and equilibrate the system before our experiment Generally in any biacore experiments
buffer should be connected and equilibrated or neither or if there is no time a minimum
of three hours of equilibration is essential otherwise when to start a new experiment do
at least six primes on the system Now that the chip is docked we will prime
the system Primly we have connected the buffer So we just say start and it takes six minutes
for the system to prime Now the prime procedure is complete we will do an immobilization of
a ligand In this case today we are actually immobilizing anti-beta two M antibody for
that immobilization Let us prepare a wizard before we setup an immobilization protocol
let us understand a little about immobilization levels The binding capacity of the chip surface will
depend on the levels of immobilized ligand The term maximum response refer to as Rmax
is described as the binding capacity of the surface in terms of the response at saturation
A theoretical Rmax value can be calculated using the formula shown below where RL is
the immobilization level and the Sm is the stoichiometric ration A theoretical calculated
Rmax is often higher that the experimentally derived Rmax for the same interaction This
could be because of several reasons such as the ligand is not fully active or that there
is steric hindrance in the interaction Different applications may require different
binding capacities and thus different immobilization levels A low Rmax is often beneficial in kinetics
analysis while higher immobilization levels are advantageous in binding analysis and concentration
measurements Today we are going to immobilize antibody on a CM5 chip using amine coupling
chemistry and the figure here shows a typical immobilization sensor gram using amine coupling The three major steps involved here are activation
of the surface esters using EDC and NHS Covalent coupling of the ligand on the sensor chip
using amine groups of the ligand and deactivation of free esters with ethanol amine we will
analyse the results of anti-beta two microglobulin immobilization later in the lecture Reference
subtraction is particularly important for assays where measurement is taken during the
sample injection The bulk contribution due to any difference
in the sample matrix and running buffer can be subtracted by using a reference surface
This reference surface is typically placed upstream of the active surface The flow cells
on the chip surface are optimized accordingly for use in pairs that is flow cell one with
flow cell two and flow cell three with flow cell four File open new wizard template identify immobilization
from surface preparation say new So we will immobilize for binding flow cell one and two
for binding and three and four for kinetics Prime before run; normalize detector
save next and the rack positions are displayed with the number of vials and the volume and
the positions As was listed on the table we will now have
a closer look on the buffers and reagents required for immobilization of anti-beta two
microglobulin The reagents include a stock concentration of anti-beta two microglobulin
from which a working concentration of 30 microgram per ml will be made using an immobilization
buffer of 10 milimolar sodium acetate pH 5 we need HEPES-EP plus pH 7.4 which will include
10 milimolar HEPES 150 milimolar NaCL 3 milimolar EDTA and 0.05 percent P20 This will be used
as the running buffer which is already connected to the system followed by priming of the system EDC and NHS in the amine coupling kit are
used in one is to one ratio for surface activation Lastly we also require one molar ethanol amine
HCl pH 8.5 for blocking the pre-ester groups on the surface We shall now proceed to use
the above mentioned reagents for our immobilization experiment We will now work on the reagents
required the immobilization of anti-beta two microglobulin on cellular chip surface We will first tell you the stock concentration
of the antibody which is 1 mg per ml of anti-beta two microglobulin In 10 milimolar sodium acetate
pH 5 to make a working antibody solution of 30 microgram per ml for this we will take
6 microliter of the ligand stock and mix it in 194 microliter of sodium acetate pH5 The
choice of the correct immobilization buffer is an important parameter to consider and
the pH scouting feature of the system will help in choosing the correct pH of the immobilization
buffer This is our 30 microgram per ml of ligand pH .. of NHS EDC and ethanol amine are prepared
and transferred to this specialized tubes used for the system We have now transferred
all the reagents into this specialized tube So we have two NHS tube Two EDC two empty
tubes for longer stability of EDC NHS which will the makes inside the system two ethanol
tube one for the blank flow cell and the other for the active flow channel and one ligand
solution We will be using HEPES-EP plus as the running buffer which will be connected
to the system prior to the initialization of the immobilization run We will now proceed to insert these tubes
into the appropriate rack and then into this system for immobilization of anti-beta two
microglobulin on the sensor chip surface Now the we made the template we will insert the
vial in the right position we just fitted them in the required volumes of different
chemicals to start with the EDC is positioned here the NHS is positioned here the ethanol
amine is positioned here and there is empty vial in a similar way another row is also
made (but) So one for the reference surface and other for the active surface to normalize
the chip normalization solution for the machine to normalize the discrepancies on the RU responses
on four different close channel We close the door this way and go on the screen
here we eject the rack we will insert the rack into the sample compartment this way
and need to lock it inside lock it inside and then go on the screen close the compartment
We go to the next tab So here there some points we need to take a before start of the run
Make sure the correct sensor chip is docked Make sure all sample reagents are loaded in
the rack and micro plate according to the rack position setup vials should be sealed
with caps place the buffer place water and make sure there is sufficient amount of water
and buffer So once we go to all these and everything
is set in the machine it shows the estimated run time as 1hour 3 minutes and we have connected
the running buffer Now we will start the experiment This will prepare the chip for the immobilization
process and immobilize the antibody or the ligand of our choice today the anti-beta two
M We need to save this as and now the immobilization process is running We will now look at the results of our anti-beta
two microglobulin immobilization performed on CM5 chip Looking at the immobilization
results dialog box there are two response levels calculated from the sensor gram The
response bound which represents the amount of ligand bound to the surface after ligand
injection whereas the response final test represents the amount of ligand covalently
bound to the surface So here as we observe 12894 RU of anti-beta
two microglobulin is immobilized on the surface of the chip Now analyzing the immobilization
sensor gram we observe the baseline here followed by EDC-NHS activation of the dextran matrix
which is again followed by baseline after activation of the surface This is followed
by covalent coupling of the ligand to the dextran matrix The buffer washes away the
loosely associated ligand molecules Deactivation and further washing away of loosely associated
ligand happens and the difference in response between these two points reflect the amount
of anti-beta two microglobulin immobilized So as we observed we have successfully immobilized
anti-bets two microglobulin and we will now proceed for our binding experiment in our
next lecture Performing an interaction analysis on active
and stable ligand surface is key to generate robust data set We have witnessed this procedure
for immobilizing anti-beta two microglobulin antibody on CM5 sensor chip surface In next
lecture we will talk about protein-protein interactions study binding analysis Thank

One comment

  1. what are the concentrations and buffer conditions of the ECD and NHS solutions? Or, if they came in a kit, what kit? How were they prepared?
    Thank you.

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