Salting In and Salting Out of proteins

Summary

This 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

• 🧬 Salting in and salting out are processes used in protein precipitation, which helps in the separation and concentration of proteins from a mixture.
• 🔬 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.
• 🌐 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.
• 🌡️ The process is influenced by factors such as pH, temperature, and the physical-chemical properties of the proteins.
• 💧 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.
• 🔄 At low salt concentrations, salts interact with charged amino acids on the protein surface, helping to stabilize the protein structure.
• 📉 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.
• 🤝 High salt concentrations cause proteins to come closer and interact with each other, leading to the formation of a precipitate.
• 📊 The solubility of proteins can be represented graphically, with solubility initially increasing with salt concentration and then dramatically decreasing at higher concentrations.
• 🧴 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.
• 🔬 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

00:00

🧪 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.

05:01

🌡️ 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.

10:03

📈 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.

Keywords

💡Salting in

Salting in refers to the process where a small amount of salt is added to an aqueous solution containing proteins, which helps stabilize the protein structure and increases their solubility. In the video's context, it is explained as the initial phase of salt addition where proteins remain soluble due to the stabilizing effect of the salt on their hydrophilic amino acids.

💡Salting out

Salting out is the phenomenon where a high concentration of salt in a solution causes proteins to precipitate out of the solution. The video describes this as the result of increased salt concentration leading to a disruption of the protein-water interactions, causing proteins to aggregate and form a precipitate that can be separated from the solution.

💡Protein precipitation

Protein precipitation is the process by which proteins are separated from a solution, often by the addition of salts or changes in conditions such as pH. In the script, it is shown as a goal of the salting process, where the desired protein is separated from the mixture for further study or use.

💡Hydrophilic amino acids

Hydrophilic amino acids are those that are attracted to water and tend to be found on the surface of proteins in an aqueous solution. The video explains that these amino acids facilitate the interaction between the protein and water, contributing to the protein's solubility, especially during the salting in process.

💡Hydrophobic amino acids

Hydrophobic amino acids are those that repel water and are typically found in the interior of a protein structure. The script describes how these amino acids contribute to the formation of a hydrophobic core within the protein, which is significant during the salting out process when the protein's solubility decreases.

💡Aqueous solution

An aqueous solution is a mixture in which the solvent is water. The video discusses the behavior of proteins in aqueous solutions, particularly how they interact with water molecules and how the addition of salts affects these interactions, leading to either salting in or salting out.

💡Ionic interactions

Ionic interactions refer to the forces between charged particles, such as the electrostatic attractions between oppositely charged ions. In the context of the video, ionic interactions are important for stabilizing protein structures, especially when salts are added at low concentrations.

💡Solubility

Solubility is the maximum amount of a substance that can dissolve in a given amount of solvent at a certain temperature. The video script discusses how the solubility of proteins is affected by the concentration of salt in the solution, with low concentrations promoting solubility (salting in) and high concentrations reducing it (salting out).

💡Solute-solvent interaction

Solute-solvent interaction describes the process by which a solute dissolves in a solvent, forming a solution. The video emphasizes the importance of this interaction in maintaining protein solubility in water and how it is influenced by the presence of salts.

💡Graph

In the context of the video, a graph is used to illustrate the relationship between salt concentration and protein solubility. The script describes a hypothetical graph where the x-axis represents salt concentration and the y-axis represents protein solubility, showing an initial increase followed by a sharp decrease as salt concentration rises.

💡Supernatant

The supernatant is the liquid that remains on top after a substance has been centrifuged or precipitated out of a solution. In the script, the supernatant is mentioned as the liquid that is discarded after the protein has been precipitated to the bottom through the salting out process.

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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