BCA method of protein estimation
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Transcript of BCA method of protein estimation
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Presented By:
Pushmeet Kaur Kohli
Bharti Nawalpuri
Lobzang
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Protein Assays
BCA Assay
Its chemistry
Reagents used
Role of reagents
Advantages and disadvantages
Applications
Comparison
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WHY?????
Necessary prior to handling protein samples for
isolation and characterization.
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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)
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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.
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Sample contains reducing agents or
copper chelating agents
Sample contains one or more
detergents ( up to 5% )
Coomassie / Bradfords Lowrys or BCA
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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.
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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
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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.
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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.
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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.
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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.)
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Solution A
Solution B Solution AB
Solution C
Solution CD
Solution D
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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
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BCA method for protein estimation
Advantages and disadvantagesApplications
Comparison
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Factorsaffecting
rate of color
formation
The types ofproteins present
in the sample
Relative amountof reactive
amino acidscontained in the
protein
Incubation time
Incubationtemperature
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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
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Interferencedue to
presence ofreducing
agent
Interferencedue to
chelatingagents
Takes sometime toproceed
Interferencedue to high
concentrationof buffer
Disadvantages
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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.
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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.
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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.
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Time
Sensitivity
Interferenceby detergents
Interference
by reducingagents
Variability due toprotein amino
acid composition
COMPARISON
advantage disadvanta
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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
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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
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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
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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.
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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
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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
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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)
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