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A full explanation about Bradford assay, Coomassie Brilliant Blue and the calibration curve

hello guys in this video I'm going to

speak about the Bradford essay

everything about Bradford assay the

principle behind Bradford essay and the

calibration curve used to measure the

concentration of proteins in a solution

so let first let's see what is Bradford

si so Bradford assay is a colorimetric

si it depends on the color of the

solution it it uses a spectrophotometer

it's a quantitative assay to measure the

protein concentration in a solution now

how does Bradford assay works work okay

the principal of Bradford si Bradford si

depends mainly on this molecule which

which is called Komatsu premium - so Kim

a cerulean blue is a very special

molecule and it has a character which is

used for the Bradford assay if that

Coomassie brilliant blue can exist in

two states okay

the first is the unstable state which is

the cationic one and the second is the

stable anionic state the very special

about canvas a brilliant blue is that

the two states don't have the same color

but the unstable state is reddish

reddish - brownish like the color you're

seeing here and the stable state is

bluish like the color you're seen here

so this is the character we use for the

Bradford assay now how does it work um

you have a protein okay you have a

solution of proteins and you want to

know the concentration of your proteins

in the solution it doesn't matter if you

have one protein or a hundred of

proteins in your solution the Bradford

assay will give you the whole

concentration of your proteins in the

solution so you have the protein

solution you take a sample of this

protein solution you mix it with the

common C brilliant blue in its unstable

state okay you mix them together now the

medium is acidic in an assay

medium now you should know that the

proteins mainly are positively charged

in an acidic medium why because of the

isoelectric point if you want to know

more about isoelectric point actually I

have a previous video the link is in the

description of this video you go to the

other video you can know more about

isolate electric point but in general

I'm telling you the proteins are mainly

put it depends but they are mainly

positively charged in an acidic medium

okay so we mix the protein and the

Kumasi brilliant blue now the colossi

brilliant blue has a lone electron pair

okay it has a lone electron pair and it

gives it can give this lone electron

pair to the protein and where exactly to

the ionizable groups so the protein is a

chain of amino acids and each amino acid

has a side chain so the protein can have

may have many ionizable groups in its

amino acid so the commercially blue can

give the protein this lone pair and in

this case when the Coomassie brilliant

blue give the protein a lone pair the

protein becomes unstable okay

because of the lone pair it becomes

unstable and then we have in the

solution and unstable Kumasi brilliant

blue and an unstable protein now the

capacitor alien blue is mainly

negatively charged the unstable protein

is mainly positively charged okay

and then these two unstable molecules

need to need they need to bind to each

other in order to be stable so they want

each other first first of all by ionic

ionic forces okay because this is

negative this is positive so they they

went together fine by an ionic force and

then they

the attachment will be further worsened

by a hydrophobic Van der Waals forces

okay why because the Coomassie brilliant

blue has a nonpolar region and the

protein the unstable protein has

hydrophobic pockets on its tertiary

structure so they can also bind to each

other by Van der Waals forces now this

this binding let's say between the

protein and the Komatsu brilliant blue

makes them to be stable okay

and when the Kumasi prelim blue is

stable it's not reddish anymore but it

turns to be bluish okay so the unstable

Kumasi beryllium blue becomes stable and

then the color becomes bluish so this is

the main principle of the Bradford si

this blue color depends on the amount of

proteins in that inside the solution so

when we have a little bit of proteins

the bluish color will be light light

blue and when we have more proteins in

the solution the blue color will be

darker so the dark is the blue color is

the more protein we have in the solution

this is the main principle of Bradford

si okay now these are the two Bradford

solution so this is the on stage so this

is the unstable Coomassie brilliant blue

okay it's reddish or brownish we can say

this is this table one it's bluish and

the blue color depends the blue color

varies from light blue to dark blue

depending on the amount of proteins it's

inside the solution okay

now this one the reddish the unstable

Coomassie brilliant blue absorb slide at

465 nanometers okay but this one absorbs

light at 595 nanometers and then we can

say that the main principle

of Bradford si8 this so the more the

solution absorbs light at 595 so the

more this solution can absorb light at

595 it means that the more bluish color

there is in the solution and which means

we have more protein the highest the

higher is the protein solution and the

higher is the protein concentration in

the solution okay this is the main

principle okay it depends on the blue

color and so it depends on the

absorption on 595 okay now how can we

measure the concentration of the protein

in the solution to measure the

concentration of the protein in the

solution we need a calibration curve and

how to how to make the calibration curve

okay we take a protein another protein

of a known concentration mainly we use

BSA so because a is a protein used to

make the calibration curve calibration

curve some people call it the standard

curve okay so to make the standard curve

or the calibration curve we use BSA

bovine serum albumin we we take a BSA

solution of a known concentration let's

say we have like two nanogram per

microliter okay and we produce several

concentrations or several delusions of

this in Kumasi brilliant blue what we

get in this so let's say this is a comma

this is comma C brilliant if this is

only Kumasi brilliant blue without any

proteins here let's say we we we add one

microliter of this solution we add two

micro liter

three micro liter four micro micro liter

and then we will have like serial

dilutions of the of the BSA protein like

two nano gram two nano gram per

milliliter four six eight we can we can

produce more we can make 10 12 okay we

can make it

as much as we want we can make one

nanogram per milliliter 3 nano gram per

milliliter we are free to to make as

much valuations as we want so if you if

you look at the color here so the Kumasi

really when the commence a brilliant

blue is alone it's like reddish to nano

gram is not so obvious the bluish color

is not so obvious then for nano gram it

becomes more and more bluish okay now we

take these a cuvette and we measure the

absorption of these solutions in a

spectrophotometer on 595 because we want

to see how much bluish color we have in

the solutions okay we use the Kumasi

brilliant blue as a blank in the

spectrophotometer and then we measure

the absorption of the other solutions

then we will get something like this a

curve so in the y-axis we have the

absorption of the solutions okay

and in the x-axis we have the

concentration as I told you we can have

as much concentrations as we want okay

here I assumed we have one nano gram per

milliliter two three four we are free

now normally the absorption should not

be more than 0.1 if you if you get an

absorption more than 0.1 then you should

dilute your protein sample more okay

because when the absorption is high has

been higher than 0.1 it it will not be

very precise so better to have

absorption less than 0.1 okay

then you will get a curve it looks

somehow like this

okay the high the higher concentration

you have the higher absorption you will

get so we don't need this we need the

linear curve okay so we get the linear

curve and we get the equation of the

curve now we have the equation the Y Y

the absorption X is the concentration so

if I have if I have a protein sample

okay let's say I have a protein sample

of an unknown concentration I take let's

say one microliter of this of this

solution I mix it with Coomassie

brilliant blue I measure the absorption

and then I say the absorption is y and

then all I have to do is to measure is

to calculate the X and then I will get

the concentration of my proteins okay

this is everything I wanted to tell you

about a Bradford si I hope you enjoyed

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