BCA method of protein estimation

8/13/2019 BCA method of protein estimation

1/31

Presented By:

Pushmeet Kaur Kohli

Bharti Nawalpuri

Lobzang


2/31

Protein Assays

BCA Assay

Its chemistry

Reagents used

Role of reagents

Advantages and disadvantages

Applications

Comparison


3/31

WHY?????

Necessary prior to handling protein samples for

isolation and characterization.


4/31

The most common methods for colorimetric detection and quantitation

of total protein are:

(Based on the chemistry involved)

ProteinDye Binding

Chemistry

(Coomassie / Bradford)

ProteinCopper

Chelation Chemistry

(BCA , Lowry)


5/31

Choice is based upon the compatibility of

the method with the samples to be

assayed.

Objective should be to select a method

requiring least manipulation and / or pre

treatment of the samples containing

interfering agents.

Each method has its own advantages and

disadvantages.


6/31

Sample contains reducing agents or

copper chelating agents

Sample contains one or more

detergents ( up to 5% )

Coomassie / Bradfords Lowrys or BCA


7/31

Best choice of the standard is the predominant protein found in the

sample.

Not always possible.

If the highly purified version of the protein sample is too expensive, then

the alternative is to use the protein which produces a very similar color

response curve with the selected protein assay curve.

The best protein choice in such cases is BSA because it is widely available

in high purity and is relatively inexpensive.


8/31

Introduced by Paul K. Smith

Colorimetric , quantitation total protein

It is compatible with samples that contain up to 5% surfactants

(detergents)

Can be performed in a test tube with reagents , resulting in apurple color which is measured at 562nm


9/31

It involves reduction of cupric to cuprous ion by the protein in

alkaline medium with highly sensitive colorimetric detection of

cuprous cation by bicinchoninic acid

The first step is the chelation of copper with the protein in an alkaline

environment to form a blue colored complex.

In this reaction, peptides containing 3 or more amino residues form a

colored chelate complex with cupric ions in an alkaline medium containing

sodium potassium tartarate.

This is referred to as biuret reaction.


10/31

This the protein copper complex formed in the biuret assay for protein

estimation.

Tripeptides and larger polypeptides or proteins react to produce light blue

or violet complex that absorbs light at 540nm.


11/31

Step 2:

Color development reaction, BCA reagent highly sensitive and

selective one, reacts with cuprous ion formed in step 1.

Purple colored reaction product forms by the chelation of 2

BCA molecules.

The BCA copper complex is water soluble and exhibits a strong

linear absorbance at 562nm with increasing protein

concentration.


12/31


13/31

Reagent A : Bicinchoninic acid (carbonate buffer and sodium tartarate)

Reagent B :Cupric sulphate solution (cupric ions)(yields a clear green working reagent)

Reagent C: Compatibility reagent.(Disulfide reducing agents, particularly DTT, 2mercatoethanol are also capable of

reducing Cu+2 to Cu+ .To minimize the affect of these copper reducers, a

compatibility agent is added to the sample before adding the BCA reagent. This

assay is also compatible with most ionic and non-ionic detergents in the presence ofdisulfide reducing agents.

Reagent D : Reconstitution buffer(used for the dilutions of BSA.)


14/31

Solution A

Solution B Solution AB

Solution C

Solution CD

Solution D


15/31

Bicinchoninic acid - 2 BCA molecules chelates with a cuprous ion,

resulting in an intense purple color

Sodium tartarate - Maintains cupric ions in solution at an alkaline pH

Carbonate buffer -Provides alkalinity to the solution for biuret reaction

Cupric Sulfate - This provides the Cu (II) ions which Chelates 4 N

of peptide bonds in protein to form a tetradentate complex (Biuretreaction)

Compatibility Reagent - It modifies disulphide reducing agents and

is added before BCA

Reconstitution buffer -Used for dilution of BSA


16/31

BCA method for protein estimation

Advantages and disadvantagesApplications

Comparison


17/31

Factorsaffecting

rate of color

formation

The types ofproteins present

in the sample

Relative amountof reactive

amino acidscontained in the

protein

Incubation time

Incubationtemperature


18/31

ADVANTAGES

Less sensitiveto types ofamino acid

present

Reagent is notsensitive to

detergents (upto 5% ) anddenaturants

Protocolflexibility is

allowed

Produces amore linear

responsecurve

Color complexis stable


19/31

Interferencedue to

presence ofreducing

agent

Interferencedue to

chelatingagents

Takes sometime toproceed

Interferencedue to high

concentrationof buffer

Disadvantages


20/31

Interference compounds:

Reducingagents :

produce highbackground

Chelators :reduces theamount of

color produced

4. High

capacitybuffers prevent

BCA fromreacting in

optimal

alkaline pH.

If samplecontains lipid,

lipoproteinand/or

biogenicamines, theabsorbance

value willincrease.


21/31

THE BETTER BCA [BCA - Reducing

Agent Compatible] Membrane protein extraction requires use of reducing agents and

detergents to maintain solubility and stability. BCA assay is compatible

with surfactants (up to 5%) but reducing agents interferes with the assay.

BCA-RAC is a modification which uses compatibility reagent which

modifies disulphide reducing agents. This assay is compatible with most

ionic and non-ionic detergents in presence of modifies disulphide reducing

agents.


22/31

BCA Protein Assay Applications

Studying protein: protein interactions

Measuring column fractions after

affinity chromatography

Estimating percent recovery ofmembrane proteins from cell extracts

High-throughput screening of fusionproteins

Highly sensitive for assaying biogenicamines.


23/31

Time

Sensitivity

Interferenceby detergents

Interference

by reducingagents

Variability due toprotein amino

acid composition

COMPARISON

advantage disadvanta


24/31

Assay absorption mechanism detection limitadvantage

s

disadvanta

ges

UV

absorptio

n

280 nm tyrosine and tryptophan absorption 0.1-100 ug/ml

small

sample

volume,

rapid, low

cost

incompatibl

e with

detergents

and

denaturatin

g agents,high

variability

Bicinchoni

nic acid

562 nm copper reduction (Cu2+ to Cu1+), BCA reaction with Cu1+

compatible with

detergents and

denaturating

agents, lowvariability

low or no

compatibilit

y with

reducingagents

Bradford

or

Coomassi

e brilliant

blue

470 nmcomplex formation between Coomassie brilliant blue dye and

proteins20-2000 ug/ml

compatible

with

reducing

agents,

rapid

incompatibl

e with

detergents

Lowry 750 nmcopper reduction by proteins, Folin-Ciocalteu reduction by the

copper-protein complex10-1000 ug/ml

high

sensitivity

and

precision

incompatibl

e with

detergents

and

reducing

agents,

longprocedure


25/31

1 )Preparation of 1x working stock solution A

Add 72ml of dd H0 with 18ml of solution A to make

1x solution A from 5X to total volume 90ml.

2 )Preparation of working stock AB

Add 88.23ml of 1x solution A into 1.77ml of solution

B in centrifuge tube to make total volume 90ml.

3 ) Preparation of working stock CDMix 1ml dd H0 with 1ml solution D, vortex and than

add 40mg reagent C in centrifuge tube ,vortex for 1

minutes

Procedure


26/31

Prepare a dilution series from BSA 2mg /ml with

solution D as follows:

BSA Conc. ( ) BSA ( ) Solution D( )

1000 150 150

750 112.5 187.5

500 75 225

250 37.5 262.5

125 18.75 281.25

75 11.25 288.75

25 3.75 296.25


27/31

Label the new test tubes according to their respective

concentration.

>Add 25 l of BSA from serial dilution into each newly

labeled clean test tubes, make a duplicates for every test

tubes.

>Add 25l of working stock solution CD ,vortex and incubatein waterbath at 37C for 30 minutes.

>Add 1ml of working solution AB into each test tubes, vortex

and incubate at 37C for 30 minutes in water bath.

>Slowly cool down to room temperature.


28/31

OBSERVATION TABLE FOR PROTEIN

ESTIMATION BY BETTER BCA KIT

S.NO Concentration

Of dilution

prepared

(ug/ml)

Volume of

various

dilutions

added (ul)

Added 25ul

of solution

CD,

vortexed

and

incubatedat 37.c for

30 minutes

Added 1ml

of solution

AB vortexed

and

incubated

at 37.c for30 minutes

Absorbance

at 562 nm

(OD) 1

Absorbance

at 562nm

(OD)2

Average

absorbance at

562nm (OD)

B0

250 0 0

125

250.011 0.014 0.0125

275

250.02 0.024 0.022

3125

250.045 0.04 0.0425

4250

250.098 0.095 0.0965

5500

250.204 0.2 0.202

6750

250.264 0.259 0.2615

71000

250.359 0.35 0.3545


29/31

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0 200 400 600 800 1000 1200

Absorbanceat562nm(OD)

concentration of BSA (ug/ml)

standard curve for protein estimation using better BCA

assay

Scalex axis 1unit =200ug/ml

yaxis 1 unit = .05o 0D


30/31

References

Smith et .al (Analytical biochemistry 1985 )

HAZEL D HILL AND JAMES G. STRAKA (Analytical biochemistry

1988)

Rhoderick E. Brown,l Kari L. Jarvis, and Kristi J. Hyland

(Analytical biochemistry 1989)

KAREN J. WIECHELMAN, ROBERT D. BRAUN, AND JIMMIE D.

FITZPATRICK (Analytical biochemistry 1988)


31/31

Documents

BCA method of protein estimation