Salting In and Salting Out of proteins
Summary
TLDRThis video tutorial from Samos Biology explores the concepts of 'salting in' and 'salting out' in protein precipitation. It explains how low salt concentrations can stabilize and increase protein solubility, a process known as 'salting in', while high concentrations lead to protein aggregation and precipitation, termed 'salting out'. The tutorial delves into the interactions between proteins, water, and salts, illustrating how these interactions dictate the solubility and precipitation of proteins, crucial for their separation and study.
Takeaways
- 𧏠Salting in and salting out are processes used in protein precipitation, which helps in the separation and concentration of proteins from a mixture.
- đŹ Salting in occurs at low salt concentrations, where the addition of a small amount of salt stabilizes the protein structure and increases solubility in an aqueous solution.
- đ Salting out is the opposite process, where high salt concentrations lead to the precipitation of proteins by disrupting their interaction with water and causing them to aggregate.
- đĄïž The process is influenced by factors such as pH, temperature, and the physical-chemical properties of the proteins.
- đ§ Hydrophilic amino acids on the surface of proteins interact with water, while hydrophobic amino acids tend to be on the inside, forming a hydrophobic core.
- đ At low salt concentrations, salts interact with charged amino acids on the protein surface, helping to stabilize the protein structure.
- đ As salt concentration increases, the interaction between water and amino acids weakens, and water starts to interact more with the salt than with the proteins.
- đ€ High salt concentrations cause proteins to come closer and interact with each other, leading to the formation of a precipitate.
- đ The solubility of proteins can be represented graphically, with solubility initially increasing with salt concentration and then dramatically decreasing at higher concentrations.
- 𧎠The process involves the formation of a 'shell' around proteins at high salt concentrations, which is hydrophilic but does not interact directly with the proteins, leading to precipitation.
- đŹ Understanding the mechanism of salting in and salting out requires knowledge of solvent-solute interactions and how salts interfere with these interactions.
Q & A
What are the two main terms discussed in the video tutorial?
-The two main terms discussed in the video tutorial are 'salting in' and 'salting out'.
What is the purpose of using salt in protein precipitation?
-Salt is used in protein precipitation to either stabilize or destabilize protein structures, depending on its concentration, to facilitate the separation and concentration of proteins from a mixture.
What is 'salting in' and how does it affect protein solubility?
-'Salting in' refers to the process where a small amount of salt is added to an aqueous protein solution, which helps to stabilize the protein structure and increases its solubility in water.
What is 'salting out' and what happens during this process?
-'Salting out' is the process where a high concentration of salt is added, causing the protein molecules to aggregate and precipitate out of the solution due to reduced solubility.
How does the hydrophilic and hydrophobic nature of amino acids in a protein affect its interaction with water and salt?
-Hydrophilic amino acids on the surface of the protein interact with water, while hydrophobic amino acids are tucked inside. When salt is added, it can initially stabilize the protein by interacting with charged amino acids, but at high concentrations, it competes with the protein for water interaction, leading to protein aggregation.
What role does the ionic interaction play in the stabilization of protein structure during salting in?
-Ionic interactions between the charged amino acids on the protein surface and the ions in the salt solution help stabilize the protein structure during salting in by enhancing the solubility of the protein in the aqueous solution.
Why does increasing the salt concentration lead to a decrease in protein solubility?
-Increasing the salt concentration leads to a decrease in protein solubility because the salt ions compete with the protein for interaction with water molecules, causing the proteins to aggregate and precipitate due to the reduced interaction with water.
How does the graph in the video illustrate the relationship between salt concentration and protein solubility?
-The graph shows an initial increase in protein solubility with the addition of a small amount of salt, followed by a sharp decrease in solubility as the salt concentration increases, indicating the transition from salting in to salting out.
What happens when proteins are precipitated to the bottom of the solution during salting out?
-When proteins are precipitated to the bottom of the solution during salting out, they form a pellet that can be separated from the supernatant, allowing for the isolation and concentration of the proteins for further study or use.
What factors can affect the solute-solvent interaction with proteins in an aqueous solution?
-Factors that can affect the solute-solvent interaction with proteins in an aqueous solution include the physical and chemical properties of the proteins, pH of the solution, and temperature.
Outlines
đ§Ș Understanding Salting In and Salting Out in Protein Precipitation
This paragraph introduces the concepts of 'salting in' and 'salting out', which are methods used in the precipitation of proteins. It explains that proteins can be precipitated by increasing the salt concentration in a solution. Initially, a low salt concentration stabilizes protein structures, making them more soluble in water, which is called 'salting in'. However, as the salt concentration increases to a high level, the proteins begin to aggregate and precipitate out of the solution, a process known as 'salting out'. The importance of understanding the interaction between the solvent, solute, and proteins is emphasized, along with the role of salt in this interaction.
đĄïž The Impact of Salt on Protein Solubility and Structure
This paragraph delves deeper into the effects of salt on protein solubility and structure. It discusses the behavior of hydrophilic and hydrophobic amino acids within a protein and how they interact with water and salt. At low salt concentrations, the positively charged amino acids on the protein's surface interact with the salt, stabilizing the protein structure and increasing its solubility. However, as the salt concentration rises, the interaction between the protein and water weakens, and the salt molecules begin to dominate the interaction with water, leading to protein precipitation. This process is further explained through the concept of a 'salting out' shell that forms around the proteins, causing them to aggregate and precipitate.
đ Visualizing the Salting In and Salting Out Process with a Graph
The final paragraph provides a graphical representation of the salting in and salting out processes. It describes a graph with salt concentration on the x-axis and protein solubility on the y-axis. Initially, as salt is added, protein solubility increases until a certain point, after which it dramatically decreases, indicating the transition from salting in to salting out. The graph illustrates the critical point at which the solubility of proteins is maximized and then precipitates due to high salt concentrations. This visual aid helps to further understand the dynamic relationship between salt concentration and protein solubility.
Mindmap
Keywords
đĄSalting in
đĄSalting out
đĄProtein precipitation
đĄHydrophilic amino acids
đĄHydrophobic amino acids
đĄAqueous solution
đĄIonic interactions
đĄSolubility
đĄSolute-solvent interaction
đĄGraph
đĄSupernatant
Highlights
Introduction to the concepts of 'salting in' and 'salting out' in the context of protein precipitation.
Explanation of the importance of salt concentration in protein precipitation and its role in either stabilizing or destabilizing protein structures.
Description of how low salt concentrations facilitate protein solubility, known as 'salting in'.
Illustration of the process by which increased salt concentration leads to protein precipitation, termed 'salting out'.
The significance of understanding solvent-solute interactions and their impact on protein structure in aqueous solutions.
Discussion on the structure of proteins, highlighting the role of hydrophilic and hydrophobic amino acids in protein-water interactions.
How the addition of small amounts of salt can stabilize the structure of proteins by interacting with charged amino acids.
The transition from protein stabilization to destabilization as salt concentration increases, leading to protein aggregation and precipitation.
The concept of a 'salt shell' or 'water shell' forming around proteins at high salt concentrations, preventing direct interaction with water molecules.
The graphical representation of protein solubility in relation to salt concentration, illustrating the points of 'salting in' and 'salting out'.
The practical applications of understanding 'salting in' and 'salting out' for protein purification and research.
The impact of environmental factors such as pH and temperature on protein solubility and the effectiveness of salting techniques.
The role of ionic interactions in stabilizing protein structures, drawing parallels to the stabilization of DNA by magnesium.
A detailed explanation of how high salt concentrations lead to the formation of a hydrophobic core within proteins, promoting aggregation.
The separation of salt and protein molecules at high concentrations, and the subsequent precipitation of proteins as a result of reduced solubility.
The methodological approach to isolating precipitated proteins for further study or application after the salting out process.
A summary of the key differences between 'salting in', which increases protein solubility, and 'salting out', which leads to protein precipitation.
Transcripts
welcome again friends welcome to another
video tutorial from Samos biology and in
this video tutorial we'll be talking the
difference between salting in and
salting out okay so you probably heard
this name earlier because I also have a
video on salting out independently so
you can see that video if you want to
study but in this video we'll focus on
the difference between salting in and
salting out okay so let's talk about it
both of these terminologies they are
used in case of the determination of
precipitation of proteins okay that
means if I draw a simple scenario it
will make you to understand let's say
the solution solution filled with
proteins see different types of let's
say this blue proteins here out there
okay so mixtures of proteins let's say
same type of proteins are concentrated
solution of proteins now in some cases
we need to have those proteins in our
hand okay so it's a mixed with solutions
and all other contents we need certain
proteins to take out from the mixture
let's say here we have another protein
that is this red one we want this blue
proteins in our hand for further
research and study so what we can do we
need to speak them up manually so how
could you do that or even if it's a
single protein mixture but still we need
some concentrated form of the protein so
if something needs to settle those
proteins down to the bottom of the
chamber or tank whatever we're using so
how will settle down the protein content
we can do that by increasing the salt
concentration or salt concentration is a
very important role in the precipitation
of proteins
okay now that salt concentration when at
very low or let us say it's very low to
low salt concentration conditions at
that time what we can do when we have
small so
to that mixture same mixture the small
amount of salt will help those proteins
to be stabilized in their structure okay
but when we slowly increase the
concentration of salt high to very high
at that condition it starts create
problem that means a very high
concentration of salt protein molecules
came to fuse with each other and
separate out from the rest of the
solution from rest of the aqueous
solution that is it's a main materialist
water obviously because it we are
talking about in the aqueous solution
okay so at very low concentration it
facilitates the solute solubility on the
other hand if we increase the
concentration it cause the protein to be
precipitated down to the bottom okay
this phenomena is known as salting um
salting out salting in means whenever we
apply a small amount of salt
concentration that helps proteins to
stabilize their structure so proteins we
may more soluble in the water for
aqueous solutions that thing is known as
salting II so this first scenario it is
known as salting II it it is
facilitating the solubility on the other
hand if we increase the temperature
increase the concentration of salt
further it will destabilize the
structure of the protein and the
proteins will precipitate down that is
known as salting out so that is the two
difference in area salting in and
salting out that we want to talk now you
know for understanding the mechanism why
salting in and salting out take place
you need to know another thing that is
the solvent solute interaction and how
this interaction is led by a protein and
water aqueous solution and how salt is
going to interfere with this interaction
so normally in the mixture
let us draw a big
mixture here with chamber in this
chamber let us say this is a protein
this protein is interacting let's say
let us talk to what see more food okay
now this proteins are interacting with
the water molecule so let us say this is
the water one they are in contact with
water molecules okay just for simplicity
I am drawing two or three things so what
happens in protein proteins are made
with amino acids right so if you look at
the structure of proteins when it they
are made with amino acids normally when
we put proteins in the aqueous solution
where water is surrounding the protein
in that condition hydrophilic amino
acids tend to predict present in the
outer surrounding sites while the
hydrophobic amino acids present the
inside of the protein okay so if you
look at the protein structure here like
this all these moieties or amino acids
that we see surrounding on the surface
area are hydrophilic that means they can
interact with water without any problem
and all the type of Hydra fully phobic
amino acids tends to fold inside
hydrophobic inside of the protein okay
and they form hydrophobic core that is
the internal core filled with
hydrophobic hydrophobic molecules cannot
interact with water or aqueous solution
that's why the thing to present inside
so this is the condition so hydrophilic
regions are involving the interaction
with water okay by a solute solvent
interaction in the solute solvent
interaction is normally depending on the
physical chemical properties of proteins
the pH of the solution the temperature
they are in these are the things they
rely on it vary
it may vary for different proteins as
the protein structure varies as a
proteins I mean acid content very we can
vary but ultimately we have an
interaction with the water molecule for
the surrounding molecules okay now once
we start adding once you start adding
the salt
in it let's say red dots are solved now
sis solved once in very slow
concentration this Sol's first start to
interact with the protein molecules
because proteins outside are charged
amino acids so they can interact with
salts to solve things to interact with
the proteins molecule at the very
beginning at very low concentration of
salt this is at very low concentration
to low concentration of salt now now as
a result what will happen
proteins have those you know amino acids
positively charged amino acids
surrounding hydrophilic amino acids
which are interacting with the salts so
this salt can help the proteins to
stabilize their structure more because
inner side they have the hydrophobic if
the outer side they get more of this
this soil so ions it will stabilize the
structure we know that you know the DNA
backbone structure is stabilized by the
presence of magnesium we know that right
these things are very important ionic
interactions now in this case they will
help the stabilization so it slightly
increase the solubility of these
proteins because this small amount of
salt concentration is not interfering
the interaction between the protein and
water is not doing any interaction so as
a result it helps in the better cellular
ization we call it as a salting II helps
the proteins to solute relies endo in
the aqueous solution now if you start
rising the concentration of the soil the
same salt we write the concentration
okay now it is becoming very high or
high and that condition it will be
filled with this salt so now what will
happen this salt component start
interacting with water right start
interacting with water that salt start
interacting with water molecules the
interaction between the amino acid and
water slowly start to become very weak
this interaction is going to be very
weak so as a result now water tends to
interact with more salts rather than
interacting with the proteins so water
is interacting with the salts and
proteins are being now alone because the
salt molecule
my teas are also going from the protein
surface so proteins are allowed round so
what proteins design at that condition
all those proteins to make the structure
even more civilized conditions the
surface area they are charged amino
acids so those protein things become
closer to interact with themselves
because there is no other one we
interact with salt is gone to interact
with water
so now proteins will come closer so what
happens here protein molecules will come
close to each other and start forming
interactions hydrophilic interactions in
the surrounding and a water molecule
along with the salt start to create this
is a salt molecules for example they
start to create a kind of shell is
called high hydrophilic sheller or water
cell that start to form surrounding
those proteins but they are not
interacting directly with the proteins
so what happens as a result as proteins
come too close to each other they start
to bind with each other they start to
interact with each other
it will turn those proteins in a huge
mass and that protein gets precipitated
down because solubility gets less and
the proteins get separated from the rest
of the solution she gets precipitated to
the bottom so once it gets precipitated
to the bottom you get the layer of
precipitates we take out the supernatant
and throw it away and we take the pellet
that is filled with proteins and we can
take our proteins in hands we can deal
with them so this is the idea of salting
out this is salting out two things
salting in we talked about salting out
we talked about now if I show you a diet
and if I show you a graph it will help
you to further understand the whole
process in much more detail let us say
here in this graph we begin with let's
let's change it a little bit it is this
once
okay let's say this is the x axis and y
axis in the y axis we add solubility
solubility of the proteins on the x axis
what you put we put the salt
concentration okay
so salt concentration increasing salt
concentration in the y axis increasing
solubility and this in this direction
okay so in this case what you will see
you will see something like that when we
start adding salt it will increase the
solubility of course single point
once that point is reached in the
solubility of salts dramatically drops
down okay so this is how the graph will
look like now in this case at the very
beginning well the Hux very little salt
concentration this is the scenario of
salting in when as I discussed earlier
though small concentration of salts
helped the structure of proteins to be
stabilized this is the scenario okay
when if I draw the protein structure it
will look something like this this will
be the proteins proteins and salts are
interacting start interacting with the
proteins something like this now the
second scenario here at this top region
where here we see the star the endpoint
of the salty knee where salt
concentration is getting to rise and at
that condition what we will get we will
get the scenario like let us say this is
the protein and the salts start to
interact with the proteins like that so
the high concentration of salt as we see
in this condition at the very end at
this point of time if you take a snap
what we will find that the soils are
separated and the professor attached
with each other
so here what we will find the image like
something like this this is a protein
and salt start
perform that that shell surrounding
their protein and proteins tend to
interact with themselves as well inside
something like that which is salting out
right so this in a sense the difference
between salting in and salting I'd so
you not forget about this idea so so I
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