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Welcome to the proteomics course. Today, we will talk about surface Plasmon resonance.
We will have a discussion on bicorn SPR technique and data
analysis. The biological systems depend upon the molecular interactions of two or more
bimolecular. So, that, they can form the stable complexes for
bimolecular interactions the principle of thermodynamics, bimolecular structure and
recognition play very crucial role. The identification of interacting protein
and protein partners of the known function with those having the uncharacterized role
by performing these type of experiment it is possible to understand the
biological process of uncharacterized proteins. The ability to screen large number of proteins
simultaneously and rapidly for biochemical activity, interactions of protein and protein,
protein lipid, protein
nucleic acids, and small molecules require various high throughput instrumentation. So
currently, the most popular methods which are use for detection of
protein-protein interactions include yeast two hybrid and protein microarrays.
As we have discussed in the previous lectures, that protein microarrays they are one of the
very robust techniques for studying protein-protein interactions, but
these microarrays require label-based detection system. Often these are fluorescence based
detection; however, the label-based detection techniques have
certain limitations, such as the tags such as fluorescent tags may interfere with the
function, including binding to the integrators and adding them to the queries
always not so straight forward. So, there is need for label-free biosensors which can
avoid these issues and allow for the real-time measurements.
So, in today's lecture, we will have a discussion on a surface Plasmon resonance. And I have
invited a guest to discuss about the bicorn technology, as well as
how to perform some data analysis using commercial software's. So, now let us discuss about surface
Plasmon resonance. This is one of the very efficient and
emerging label-free techniques for studying bimolecular interactions. The surface Plasmon
resonance biosensors or optical sensors which can exploit the
surface Plasmonpolaritons the surface electromagnetic waves that can propagate parallel to a metal
or dielectric surface. SPR is used to probe interactions
between an analyze in solution and a receptor that are attach to the SPR sensor surface.
Binding of molecules in the solution to the surface immobilize
receptors changes the refractive index of the medium near the surface.
The change in the refractive index of medium can be monitored in real-time to measure accurately
the amount of bound analyze it is affinity for the receptor
and the association and dissociation kinetics of the reaction. Over the past decade the
SPR biosensor technology has made significant advancement and a large
number of SPR sensor platforms. The bimolecular recognition elements and measurement formats
have been developed. The major strength of SPR
biosensors are their versatility and ease of use, the SPR allows the analyses of receptor
ligand interaction for wide range of molecular weights, affinities, and
demonstrates the compatibility for small molecules and other chemicals. The SPR biosensors have
played a very important role in biological research into
bimolecular and their interactions. And now they are increasingly being use for detection
and identification of chemical and biological interactions.
Now, let us move on to the bicorn SPR technology. So, there has been growing interest in commercialization
of SPR biosensors, which has lead to a number
of systems available in market. The commercial instrument from Pharmacia and then bicorn
became available in 1990. Bicornare optical biosensors which can
be used to monitor macromolecular interactions in real-time, without need to label the bimolecular.
The bicorn is a versatile platform to determine the kinetic
rate constant for a variety of interactions. A number of commercial SPR biosensor instruments
are available since then; however, the bicorn system is still
dominate this field. So, let us now discuss about the bicorn technology with Lilith kishore
the business leader research products in g e healthcare life sciences
who handle the bicorn system Hello
Hi This pleasure to introduce Mr Lalith kishore
from g e healthcare to discuss about bicorn surface Plasmon resonance technology. Lalith
kishore is business
leader research product in g e life sciences and today we will discuss about the surface
Plasmon resonance technology and it is various applications in
proteomics. So, Lalith welcome to the discussion on SPR technology
Thank you thank you So, how long have been associated withbicorn
SPR technology with g e healthcare. I have been with g e healthcare for the last12
years and been associated with bicorn for the last four and half years, been associated
with proteomics for a
slightly longer time for about 7 years now. So, can you just brief us about your experience
of using the biocore technology from last several years my educational background is
that I am not a biologist Though bicorn is a largely biological tool
my background is in chemical engineering. So, I am a chemical engineer with MS and management;
however, I find
very very disuses for SPR technology these days, I find its use in a biopharmaceuticals,
I find its use in basic research, in nanotechnology, in pharmaceutical
industry, in Q Clabs. So, basically I think it is my chemistry and chemical engineering
background that helps me work with work with bicorn and quite a lot of
applications that I see across the country on bicorn technology.
So, you are utilizing your vary background to apply on the different biological problems.
Yes yes that is what I think SPR technology also does that actually,
uses a very simple technology and applies it to different things in biology and actually
gives out the results that are therefore, everyone to see. So, Lalith can
you please tell us, how you got interested in working on the surface Plasmon resonance
technology. Yes g e healthcare has been associated with
bicorn, as a company for a very long time. In the year 2006 we actually acquired the
bicorn. And up until that
bicorn was there only in some places in India. So, I got into bicorn in 2006 out of interest
in chemical interactions and interaction analysis, and ever since I
have been just working on biacore and label-free interaction analysis technologies. So, can
you mentioncurrently what are the major applications of SPR in the
area of proteomics? Actually very vast very wide ranging applications
of SPR starting from a simple binding analysis or kinetics analysis, analysis of affinity
of interactions whether
it is protein-protein interactions, protein DNA interactions, protein RNAinteractions,
protein small molecule interactions. In drug discovery quality control
varied applications of SPR actually we support very large variety of customers from different
backgrounds who want to do SPR in their labs. So, definitely
you can see probably that SPR application will be very broad in almost all the proteomics
laboratory depending upon their experiments and that Question they
want to ask... So, biacore is one of the pioneer in the field
of studying the label-free interactions and studying about bimolecular interactions. Can
you tell some of the latest
advancement what are the major applications by using the biacore technology. Let me start
with a video That shows basically what SPR is and then
probably followed by another video of how biological analysis happens on biacore, and
then just a few videos shot
once on how biacore works. And then I quickly come to the applications of biacore.
So we can...Let me show you the first video.
So, what I show here is the basic SPR phenomenon right. This is the SPR chip which you see
here, on the top of SPR chip is a gold layer, and on the top of it
is a flow cell at the bottom you see a hemispherical prism. So, SPR phenomenon is pretty simple
that when you actually have the prism and you shine a laser
light through the prism, as you will see shortly. When you shine the laser light through the
prism the light reflects at a total angel of total internal reflection. The
light reflects at the angle of total internal reflection and evanescent energy waves are
created on the top of the chip. And these evanescent energy waves are
also called as surface Plasmon's. And this surface Plasmon's are the once which have
used to actually study biological interactions. When I go to video two
now you will see how biological interactions are studying.
Let us assume that you have an interaction a plus b gives a b. What you do in SPR is
you take one of the interact antssay b, and put it on the chip and pass a
over it. Let me show you how it happens. You take one of the interactants which is b in
this case, and you actually immobilized it on the chip, you can see the
molecules getting immobilized right now. And then when the molecules get immobilized there
is an increase in mass which changes the refractive index and
that is measured in real-time. Now you pass the second interact ant a over it, if binding
happens and a b gets formed you see a further increase in mass which
is again measure in real-time. You stop the flow of a and start flowing a buffer it comes
off in a dissociation and that dissociation is also seen in real-time. So,
essential what you are doing with biacore is actually just measuring the amount of mass
on surface of the chip. The mass on the surface increases or decreases
and that increase or decrease is measured in real-time. This phenomenon is what we apply
now to study biological interactions actually, biacore is b i a for
biological interaction analysis So, let me show you a few videos examples,
of how biacore can be used to study some in some experiments. This is an example, where
a plus b gives a b, if
you see the curve and if the curve is existing it means, a b is formed. If you pass a over
b and there is no response which means, if you see a flat line then a b
is not formed.
So, it is a very simple example, where you can actually decide whether the interaction
is happening or not happening. I will show you another example now of
kinetic analysis where, you will see two examples. The first example will be that of an extremely
rapid association. So, you see this slope of the curve goes up
very fast and comes down extremely fast. So, this is a rapid association and dissociation
whereas, if you see the slope slope of this curve that is about to come
up it is very slow association and very slow dissociation. So, just by looking at these
curves you are able to actually tell if the interaction is fast or if it is slow.
So, these are some of the examples of biacore technology at work. I think unlike microarrays
were people can detect the interaction, but they cannot tell the
nature of the interaction here, the edge is that by looking at this type of kinetics in
the curves. One can also tell about the type of association
dissociation and the Overall kinetic analysis.
So, and here I have on on myp p t a very simple analysis of different things that can be done
with biacore. So, once someone ask me what can be done with
biacore, these are the 6 things that can be done with biacore. So, very shortly put these
are the 6 applications of biacore. Whenever a plus b forms a b when
you are studying an interaction a plus b gives a b. The questions you ask is a first question
you ask is does the interaction happen or not which means, is the
molecule a b formed or not. The second question you ask is how fast the interaction is or
how slow is the dissociation. The third question that you ask is how
strong the interaction is. So, what is the affinity of the interaction? The fourth question
you ask is how much of the analyze is there Which means, what is the
what the concentration of this is analyze. Sometimes in drug industry you ask is this
interaction is safe or interaction is not safe.
And sometimes if you have hydrogenanalyze and you see the binding is happening you want
to ask what is it that is binding, because there are too many
components in this analite. So, what is it specially binding and these are the five different
things that you can do with biacore. So, I think you rightly mentioned
identifying the very specific interact or is the most challenging aspect of it, yes,
because that is why many time people fail and they discover the false
interactors. So, I think that is where the SPR has edge over conventional techniques
like immunoprecipitation or yeast two hybrids and some other other large
screening methods where there is the good chance of identifying the false positives.
Sure sure there is a lot of promiscuous binders in screening experiments.
Which will be avoided if you this specificity that biacore gives you?
So, we start with each of these applications in details. The first application which we
will talk about is this specificity application. Now some interesting
questions that are asked when you when you are doing specificity applications. Is the
drug binding to the receptor or not, is the map by identifying a strain or
not, or is there any nonspecific binding in the interaction that I am studying, and these
questions are very easily answered by biacore.
Shown here in my p p t is actually an example, where we are looking for binding and here
someone came to ask with about 40 different compounds and they
want to see if any of these compounds binds to a receptor. And here we have forty of these
experiments done, most of these are not binding or binding at a
very base level, but if you look at this presentation. I highlighted one spot here with one molecule
that a shown circle in red, this particular molecule is actually
binding to the receptor. So, at the end of a very short experiment of looking at the
receptor versus candidate binding, you are able to determine which of these
candidates is actually binding to the given receptor a very simple example of specificity.
Going on to the next application, so, I will interrupt you here. So, basically they just
demonstrate that even if we are very much unaware about the components
which could be interacting, this could be a good screening tool right, because if there
is a real strong interaction and specific interaction then probably we can
see some out like this if you want to do that first level screening to just quickly find
binders. In some kind of a screening experiment like you mentioned then
this would be a very good starting point for you to quickly find those binders and take
them to the next level. Many times when we have a thirst of identifying
some like large drug library right. Or small compounds and then at that time maybe to begin
with good straining tool. Yeah I would also like to point out that this
a very small experiment, it barely takes about one minute to this experiment. So, basically
per minute you can do
one screen and that way you will have a lot of compound screened, you know you know very
rapid manner. So, that is the first step in the specificity
experiments. Now, let us assume that you found this candidate
and you think that it is a specific binder and now you want to look at the kinetics of
the interaction. And
everyone understands that kinetics is an extremely important part in drug discovery, in proteomics
when you look at interactions one of the important things
that you need look at is what is the on rate and off rate of the interaction.
And so, in that sense how fast is this interaction happen and which candidate is kinetically
preferred, because if you have two candidates both of them trying to
be drugs you should choose the candidates that is kinetically preferred. And in most
cases in recent cases actually kinetics is being used to shows similarity of
drugs for examples, there is a lot of biosimilars coming out of India. And biosimilars manufacturers
actually want to show that their molecule is similar, or
equal to the innovators drug then one good way of showing that their similar, is by showing...
So, that is where again kinetics experiments will be helpful to you experimenters scientists
can actually calculate the k on k off, or K a k d and the k capital d
which is the affinity of the interaction. They can calculate this and in a very fast
way actually understand the interaction a little better than they did before,
because in the first instance they only knew whether the interaction was happening or not.
Whereas right now they also know that the what the kinetic
parameters of this interaction are. And once they know the kinetics of the interaction
they come to affinity. So, identifying the on rate off rate and dissociation
constant I think this provides a very strong tool and information by... For characterization,
For characterization and that is i think the big thirst of all the
pharmaceuticals doing by biosimilars. Anyone who is pursuing biopharmaceuticals
or recombinant proteins by biosimilarsor even novel drug discovery people in the small molecule
arena. One to
actually characterize the interaction in terms the in terms of the on rate the off rate and
the affinity of the interaction. And that something that is very well
accomplished by by the SPRtechnology. So, when it comes to affinity as you can see here,
how strong is the bond. Is the binding strong enough to be
physiologically important and this is one very important thing, because more and more
drugs are coming out these days which are one dose a day drugs or
which are you know fast acting drugs. And these kind of discoveries depend a lot
on kinetics and affinity of the interactions. And that is where biacore can actually come
in in a very big way and
help people you know genetically or protein engineer their drug. So, that they actually
perform better than existing drugs. For a good comparison with the
existing to make drugs better Or to discover novel drugs they are actually act better.
Both of the cases I think SPR technology can be useful.
Once you know the affinity and concentration and I always think that concentration is very
less studied, but it is very important is, because if you look at
concentration analysis across the world for proteins. There is no way that someone can
measure active protein concentration without having calibrants in their
hands. Whenever you give a student a protein and
say please measure the concentration the first thing that he will ask you is for calibrants.
He will ask you for
standards and the problem with these standards is that sometimes they are not available,
sometimes they are extremely expensive, and being proteins like you
know sometimes they are not stable. They are not stable. So, there is a great
need for having a concentration analysis technique that does not need calibrants. And that is
where biacore comes in
againbiacore does something that is called CFCA which is calibrant free concentration
analysis. And. So, within 5 minutes if you have a specific binder for a
protein you can actually calculate the concentration of that protein without the need for the calibrants.
And since you already seen in screen one, where we
talked about specificity we are talking about specific binding. So, what is measured is
not just total protein concentration. What is measured is specific active
protein concentration. I think it is very strong application of it, because many times
you would like to know how accurately you can determine the protein
concentration. Absolutely especially in quality control and
in filling in in biopharmaceuticals again where people need to exactly estimate how
much they are actually filling in
the final vial. That actually goes to the patient they need more accurate methods of
measuring active protein concentration.
And that is where I think biacore will play a very big role in letting people estimate
that active protein concentration. Now once you seen concentration the
next thing is immunogenicity. Worldwide with the increased biopharmaceuticals drugs everywhere
needs to be tested for immunogenicity. Immunogenicity is about direct measurement
of antidrug antibodies, in which which should be measured in serum. It is also about bringing
a regulatory
framework into a system right we talked about only technology and science, but suddenly
when it comes to drugs regulators come in. So, can we actually accurately confidently
measure antidrug antibodies in animal sera or human sera, at clinical trial levels? And
biacore can actually be used
for accurate measurement of antidrug antibodies and for immunogenicity testing of biopharmaceuticals.
So, that is another major application of biacore as it
comes. The last application of biacore, which if you remember when I talked about in the
first slide about specificity. Now if you have a heterogeneous
mixture that is flowing over a ligand and something within that mixture is actually
bound. And you see a curve and you know that something has bound, but
you do not know what it is that is bound. Binding. So, you can use the technique called
SPR MS where you can take the bound analyte separate it into a vial
and then actually take to a mass spec and identify the protein. So, in addition to doing
all that they did before, now you can actually also find out what is it that
is bound. So, definitely it is very important, because many times you will not know like
what the unknown target is which is interacting or binding right. Yes.
So, if you do not if you have this unknown for example, if you have a receptor and if
you have cell lysate or if you have some kind of a homogeneous tissue
lysate which we you have flowing over the ligand and now you can actually find out what
that proteins are. So, it is used in applications like ligand fishing for
example, where you have fishing for a ligand. So, those are the kind of examples that...
But in MS you will definitely required protein above certain threshold right. So, how you
overcome that issue, because the binding will minimal. And of
course...So, one thing is that this is a small interaction happening. So, the amount of protein
that you collect may not be sufficient the only way you can
overcome it is by actually doing it multiple times collecting enough. So, that you can
actually get some kind of an MS response and that is what most of our
users do is that they run the same binding as say about 10-20 times.
And collect the bound analyte and then take it to an MS and then get their result. So,
in biacore you have a way to collect the flow in the flow cell. And then
actually you can accumulate that in multiple multiple runs and then take it. Then, concentrate
that and then you do further mass spec. Correct.
Yes. So, just to summarize again I will just go back to my the first slide that I showed
here, which is here. This are the are the six things that biacore can be
used for specificity, kinetics, affinity, concentration, immunogenicity, and mass spec
analysis. So, that is those are the 6 broad applications of a biacore SPR
technology. That is what very important, because many
times a good decision about these products before hand about characterizing these proteins,
can save you lot of
money lot of efforts down the road, because... Absolutely. If if you can do those experiments
in the beginning most often when we actually talk to pharma
industry, you know we tell them that biacore or SPR technology is not for success, but
more for failure. Only thing is that we say that it is for early failure and
it is for cheap failure. So, do not spend too much money on something that does not
work, might as well fail early. So, that is the basic reason why SPR should
be used. I think that is very important. So, can you briefly tell us about what are the
major instrumentation
available from the biacore technology currently for doing the surface Plasmon resonance based
experiments?
There are basically four different biacore instruments available, there is a very small
biacore which is called the x 100.You can see it on pitch it has two flow
cells, and it can do some beginning analysis, and then you have the biacore 3000 which is
an academic favorite a lot of academicians like it, lot of lot of
customers in India who are academics have the biacore 3000. Then we have the latest
which is the biacore t 200 very special again, because it has all the things
that the biacore 3000 has, but it is the regulatory approved. So, if you are company that works
with f d a that works with that works with you know d g c i or
some of these regulatory authorities, then I think that we should be using the biacore
t 200. The biacore4000 has 20 different immobilization sites on it and
biacore4000 can be used with multiple 384 well with robotics.
So, if you are company that has you know extremely high throughput screening, if you are company
that does a lot of immunogenicity experiments or if you
are doing batch testing or release testing using biacore then you should be using the
biacore4000. I must say that most of the customers in India use either the
biacore three thousand or use the biacore t 200. So, how easy it is to do these SPR
experiment and especially the kinetic analysis by using some software
available from biacore. The the most important thing in a biacore
experiment is actually, the experiment design. It is very easy to do the analysis and let
me just show you an example
of a typical result. And let me show you how the analysis works out. So, if you can see
my computer. So, I am going to open a typical biacore result and here
is a biacore result, this is a biacore result where5 samples of different concentrations
We run over a fixed ligand which was on the chip. And now I am trying to dokinetic analysis.
So, if you look at these by results, I select them and I show the
results and these are the results. Now this is what are typical results looks like and
there is nothing to be worried about, because it looks odd the idea is that
your results are all embedded somewhere here. These two big peaks that you see are regenerations.
So, the first thing that I do when I do the analysis is
actually select the regenerations which are do not need and cut them out.
So, I cut them out here. So, I say cut and then the rest of the result. So, you want
adjust your response access. So, now I have 5 different concentrations
1,2,3,4,5 in different colors. And you have this is the association face, and this is
the dissociation face. Now I am going to just baseline this result.
So, I select the baseline here, and then I just go to the adjustment of y axis and I
say 0 at the average of selection and then I say add his name. And now all my
five results are shown here. Now I am going to do a quick kinetic analysis and it is really
extremely simple to do a kinetic analysis, because all that I do is say
calculate. I say kinetics simultaneous k on and k off. And I have already done the cutting
and the y transformation. So, I say next and then if I want to I can go
and adjust the start and the end time. So, I can actually move this to adjust the start
time and the end the time of association and dissociation which I
sometimes do, but I think this is pretty well picked by the software already. So, I do not
need to do much, I say next and I enter the different concentrations of
each of the samples which were run they are entered here now this is very important. These
concentrations are known concentrations which which you ran
already ,which you are you actually made the concentrations of the analyte and then ran
over the ligand. So, you would do that. Now one of the things
that is important here is you choose the model. So, preferably it is always better whenever
you do
characterization the more you know about your system the better characterization results
you get. So, in this case suppose you do know that it is a one to one
binding then you would choose that binding, but if you want to you can actually change
the the binding model it could be a bivalent analyte it could be a
bivalent ligand.
It could be a heterogeneous ligand. So, depending on the model you choose. So, you choose the
model that you want to here. And then you say fit and what
happens is curves get fit and there is also thrown up. So, your k on and k off are displayed
here. So, it is as simple as that. So, you all you need to do is take a
ligand immobilize it on a chip. Run about 5 different concentrations of your
analyte over the chip and this simple analyte and each of these results if you look at the
x axis carefully, it is0to
600; that means, each one of these runs the entire run was 10 minutes. And you ran 5 samples.
So, it is 5 into 10 minutes. So, 50 minutes with the time taken
in between the runs about another 10 to 20 minutes about one hour 10 minutes 1 hour 20
minutes you have the results and you have already characterized
your results.
Because you have the k on and k off calculated. And you can also quickly check how good your
results are by quickly checking the residuals and you can see
the residuals here the chi square values are really between minus 5 to plus 5 percent.
So, extremely good. Many times fitting reactions and a very fast analysis
that gives you k on and k off I think it is very extremely fast and very good, very easy
to use... Interactions. Very easy to use software for your analysis.
So, it is very useful and informative to see the analysis like how easy this to perform
the kinetic analysis. Can you give some specific example, of doing kinetic
analysis by using biacore system? Let me show you one example, and this again goes back
to why kinetics is important. Kinetics is extremely important in
biacore analysis and let me show you an example. So, here is a pitch about three slides on
kinetic and affinity analysis using biacore. So, if you look at this
slide.
Now, here on this slide you have three interactions which are captured you have interaction one
which is captured in blue, interaction two which is captured in
red, and interaction three which is captured in black. The important thing about all these
three interactions is that all these three interactions have the same
affinity. They have varying k on and k off, but they have the same affinity and this is
important in drug discovery, because let us say for example, you are
looking for pain relief, if you want pain relief you want a drug that acts fast and
that stays on for a very long time, but if you want a sleeping pill for example,
you want a drug that actually acts slowly, but stays on for only a reasonable amount
of time and comes off fast enough. So, kinetics is very important in choosing
a drug candidate now this is an example where if someone chose just on the basis of affinity
all these three would
have been the same, but since they would make their choice based on kinetics they can actually
decide based on thek on and the k off now here is a real life.
Example in the next slide which I show you which is a publication from Amgen and from
Davidmyszka of university Utah. Where actually Amgen uses this
data directly to do their clone selection. So, they are doing maps selection of maps
selection of clones for maps, and if you look at this say clone number one,
and clone number two have similar, affinities, but you know if you look at their on rate
and off rate there is a ten folded difference. So, if you look at these two
clones for example, which clone should they go for, they should go for the clone which
is having more kinetically relevant properties. If they use only affinity
for making that choice then there is no choice at all they are both the same. So, this is
a live example of where kinetics data is actually being used to capture
information regarding k on and k off and then make a more educated decision, knowledgeable
decision on which map to go forward with, correct. Now this is
very interesting to see this, because you will feel that if you just rely totally on
the k d values right if you do not go Individual on the k on and k off. Yes. So,
distribute that k d value into k on and k off and make a more knowledgeable eyes.
So, it is very interesting example to appreciate the power of kinetic analysis right. Can you
just brief us about what are the major limitations or shortcomings of
various surface Plasmon resonance based technologies. Absolutely. So, like any technology this technology
should not be viewed as a silver bullet. It does have
short comings one of the significant shortcomings being that, if there is any structural differentiation
in the protein this would not be able to capture it is mass
based sensor. So, any structural changes will not be captured, there is also a problem that
there are situations where you are unable to immobilize the protein
that you have on to the chip. So, you might have to use some capture techniques
to do that. There is also there is big question about you know. So, you answer these questions
about
interaction you answer questions about whether the interaction happens or not how fast how
slow how strong how much. You say whether it is safe or not
with immunogenicity and you know what is binding, but then sometimes you want to ask the question
why is it binding And why is it binding is answered by structural
studies or thermodynamic studies. So, that is where I think biacore can give you a little
bit of a direction, but I
think you should do more of you know an n n m r study or you should do microcalorimetry
and that is what will probably give you more answers on why the
interaction is happening. So, further in depth studies will be required on those. Interactions.
So, when you have an interaction happening and when it is happen
when it is happening very fast. Now, why is it happening fast is transition
state thermodynamics. So, you will have to ask questions in thermodynamics to get those
answers. So, sometimes
further study is essential and biacore kind stops with these applications. Would you have
any final advice take home to the. So, here is here is basically three
rules that we have in biacore. The first rule in biacore experiments is biacore technology
is extremely easy to understand, biacore technology is extremely easy
to analyze, a lot of time must be spent on experiment design. The first thing that I
would do if I were working with biacore is spend a lot of time on very
carefully considered experiment design. The second thing that I would do if I were conducting
a biacore experiment is that I would make sure that I have an
extremely pure ligand that I will put on the chip and that is extremely important. The
last thing that I would consider and this is true of all experiments and I
would agree with me is that it is garbage in garbage out so, make sure your sample preps
are correct and remember that this is an analytical instrument, as
unlike many other techniques in biotech, this is an analytical instrument it is a mass sensor
at the end of the day you could call it in extremely sophisticated
weighing machine. So, if you put something on it it will give you the weight, it is as
simple as that only thing is you have to do it right. So, I would say that
make sure that your sample prep is perfect your experiment design is perfect make sure
your ligand is pure. So, I think you very rightly mentioned that
a good experimental design identifying the good ligands working out the chemistry for
immobilization. As well as
doing the very well sample preparation. All of these are very essential component for
doing any successful proteome experiment, and especially the surface
Plasmon type of experiments. So, thank you very much Lalith fordiscussing about the biacore
technology with us today Thank you.
And I hope it is informative and useful for my students. Thank you thank you
So, after having a discussion with Lalith kishore on biacore SPR technology. I hope
now you are very clear about the instrumentation, the various properties
which can be studied by using the biacore system and how quickly one can perform the
data analysis. So, conclusion from this whole discussion is that, since
the introduction of the biacore SPR instrument. The SPR spectroscopy has become widely used
from chemistry and biochemistry to characterize biological
surfaces, and monitor the bimolecular binding events. Overall the success of SPR technique
is due to the following factors. First the kinetic measurement in
real-time that is the major strength of data obtained from the SPR instruments. Second
monitoring the adsorption of unlabeled analyzes molecules to the
surface. And third its ability to monitor weakly bound interactions due to high surface
sensitivity of SPR biosensors.
So, in summary in today's lecture, you have learnt about significance of studying the
bimolecular interactions, how surface Plasmon resonance technique can
be used for studying the bimolecular interaction. A brief introduction of biacore technology
and data analysis and then certain issues and challenges which are
associated with SPR technology. So, we will continue our discussion on label-free techniques
in the subsequent lectures. Thank you.