TOTAL PROTEIN ASSAYS

Background
Bradford Assay Procedure
References
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Background & Theory
Four spectroscopic methods are routinely used to determine the concentration of
protein in a solution (1). hese include measurement of the protein!s intrinsic "#
a$sor$ance and three methods %hich generate a protein&dependent color change' the
(o%ry assay ())* the +mith copper,$icinchoninic assay (3) and the Bradford dye assay
(-). Although one or more these methods is used routinely in almost e.ery $iochemical
la$oratory* none of the procedures are particularly con.enient* for the reasons descri$ed
$elo%.
he first* "# a$sor$ance* re/uires that a pure protein %ith kno%n e0tinction
coefficient $e used* in a solution free of interfering ("# a$sor$ing) su$stances. As an
appro0imation* the protein concentration of a solution can $e estimated $y using either
of the follo%ing e/uations'
A
)12
3 1 A (m(,cm mg) 0 45onc.6 (mg,m() 0 1 (cm)
A
)25
3 31 A (m(,cm mg) 0 45onc.6 (mg,m() 0 1 (cm)
7ifferent proteins* ho%e.er* ha.e %idely different e0tinction coefficients at $oth )12
and )25 nm* and concentration estimates o$tained this %ay should $e .ie%ed %ith
considera$le skepticism. Again* this assay re/uires that the protein solution $e free of
other "# a$sor$ing su$stances* and that the measurements $e made using a /uart8
cu.ette.
he (o%ry and copper,$icinchoninic assays are $ased on reduction of 5u
)9
to
5u
19
$y amides. Although this makes them potentially /uite accurate* they re/uire the
preparation of se.eral reagent solutions* %hich must $e carefully measured and mi0ed
during the assay. his is follo%ed $y lengthy* precisely timed incu$ations at closely
controlled* ele.ated temperatures* and then immediate a$sor$ance measurements of the
unsta$le solutions. Both assays may $e affected $y other su$stances fre/uently present
in $iochemical solutions* including detergents* lipids* $uffers and reducing agents (1).
his re/uires that the assays also include a series of standard solutions* each %ith a
different* kno%n concentration of protein* $ut other%ise ha.ing the same composition as
the sample solutions.
he Bradford dye assay is $ased on the e/uili$rium $et%een three forms of 5oomassie
Blue : dye. "nder strongly acid conditions* the dye is most sta$le as a dou$ly&
protonated red form. "pon $inding to protein* ho%e.er* it is most sta$le as an
unprotonated* $lue form.


Protein
Red ;3< :reen ;3< Blue ;3< Blue&Protein
(-=2 nm) (>52 nm) (5?2 nm) (5?2 nm)
@
9
@
9
he Bradford assay is faster* in.ol.es fe%er mi0ing steps* does not re/uire heating*
and gi.es a more sta$le colorimetric response than the assays descri$ed a$o.e. (ike the
other assays* ho%e.er* its response is prone to influence from non protein sources*
particularly detergents* and $ecomes progressi.ely more nonlinear at the high end of its
useful protein concentration range. he response is also protein dependent* and .aries
%ith the composition of the protein. (+ee e0amples) hese limitations make protein
standard solutions necessary.
he reagents for all of the protein assays are %idely a.aila$le* and pre&%eighed
reagents (5)* reagent mi0tures (5)* and reagent solutions (>*=) are a.aila$le for the
assays. Aodifications of these assays* using proprietary solutions (1) or different
protocols or formats are also descri$ed in the literature (?) or are commercially a.aila$le
(12).

BRADFORD PROTEIN ASSAY PROCEDURE
Reagent!
Dye tock & 5oomassie Blue : (5.B.C -)>55) (122 mg) is dissol.ed in 52 m( of
methanol. (Bf tur$id* the solution is treated %ith Dorit (122 mg) and filtered through a
glass&fi$er filter.) he solution is added to 122 m( of 15E @3PF-* and diluted to )22
m( %ith %ater. he solution should $e dark red* and ha.e a p@ of &2.21. he final
reagent concentrations are 2.5 mg,m( 5oomassie Blue :* )5E methanol* and -).5E
@ 3PF-. he solution is sta$le indefinitely in a dark $ottle at -G5.
Aay reagent & he assay reagent is prepared $y diluting 1 .olume of the dye stock
%ith - .olumes of distilled @
)
F. he solution should appear $ro%n* and ha.e a p@ of
1.1. Bt is sta$le for %eeks in a dark $ottle at -G5.
Prote"n Standard & Protein standards should $e prepared in the same $uffer as the
samples to $e assayed. A con.enient standard cur.e can $e made using $o.ine serum
al$umin (B+A) %ith concentrations of 2* )52* 522* 1222* 1522* )222 Hg,m( for the
standard assay* and 2* 12* )2* 32* -2* 52 Hg,m( for the microassay.
Standard Prote"n Aay Procedure #$%% & $%%% 'g()L *rote"n+!
Prepare si0 standard solutions (1 m( each) containing 2* )52* 522* 1222* 1522 and )222
Hg,m( B+A.
+et the spectrophotometer to collect the spectra o.er a %a.elength range from -22 to
=22 nm and o.er an a$sor$ance range of 2 to ) A$sor$ance units* and to o.erlay the
collected spectra.
"se a - m( plastic cu.ette filled %ith distilled %ater to $lank the spectrophotometer
o.er this %a.elength range
Impty the plastic cu.ette into a test tu$e and shake out any remaining li/uid. hen
add'
).2 m( Assay reagent
2.2- m( of protein standard solution* starting %ith the lo%est protein
concentration and %orking up* or one of the samples to $e assayed.
5o.er %ith Parafilm and gently in.ert se.eral times to mi0
Record the a$sor$ance spectrum of the sample from -22 to =22 nm * and note the
a$sor$ance at 5?5 nm.
Repeat the steps a$o.e for each of the protein standards and for the samples to $e
assayed.
I0amine the spectra of the standards and samples. Bf any spectrum has an a$sor$ance
at 5?5 nm greater than )* or if any sample has an a$sor$ance greater than the greatest
a$sor$ance for any of the standards* dilute the sample $y a kno%n amount and repeat the
assay. At one %a.elength* appro0imately 5=5 nm* all of the spectra should ha.e the
same a$sor$ance. (+uch an intersection is called an isos$estic point and is a defining
characteristic of solutions containing the same total concentration of an a$sor$ing
species %ith t%o possi$le forms.) Bf any spectrum does not intersect the other spectra at
or near the isos$estic point* it should $e adJusted or reJected and repeated. (Bt is
sometimes possi$le to adJust the $aseline of a spectrum $y determining the difference in
a$sor$ance at the isos$estic point from the a$sor$ance of the other spectra at that
%a.elength* and adding the difference to the a$sor$ance .alues at e.ery %a.elength of
the spectrum. his correction %orks $est at %a.elengths close to the isos$estic point*
and re/uires some discrimination $y the spectroscopist.)
Prepare a graph of A$sor$ance at 5?5 nm .s 4Protein6 for the protein standards.
I0amine the graphed points and decide if any should $e reJected. (Fften a single
point can $e reJected %ithout in.alidating the standard cur.e* $ut if more than one point
appears /uestiona$le the assay should $e repeated.) he Bradford assay gi.es a
hyper$olic plot for a$sor$ance .ersus protein concentration* $ut %ithin a range of
relati.ely lo% protein concentrations* the hyper$olic cur.e can $e appro0imated
reasona$ly %ell $y a straight line. "se a $est&fit straight line to fit the points if you feel it
%ill gi.e a good fit. Bf not* dra% a smooth cur.e that falls on or near each of the data
points.
o determine the protein concentration of a sample from it a$sor$ance* use the
standard cur.e to find the concentration of standard that %ould ha.e the same
a$sor$ance as the sample.
,"croaay Procedure #-.% 'g()L *rote"n+!
Prepare fi.e standard solutions (1 m( each) containing 2* 12* )2* 32* -2 and 52
Hg,m( B+A
o a 1.- m( plastic cu.ette* add'
2.) m( 7ye stock
2.1 m( of one of the protein standard solutions or samples to $e assayed
(containing ;122 Hg of protein for ;52 Hg,m( standards)
5o.er %ith Parafilm and gently in.ert se.eral times to mi0.
Follo% the procedure descri$ed a$o.e for the standard assay procedure.
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REFERENCES
1. +toscheck* 5. 1??2 KLuantification of ProteinK Methods in Enzymology * /0$!52&>1.
). (o%ry* F. Rose$rough* A.* Farr* A. and Randall* R. 1?51 J. Biol. Chem . /12!)>5.
3. +mith* P. et al.* (1?15) Anal. Biochem. /.%!=>&15.
-. Bradford* A. 1?=> KA Rapid and +ensiti.e Aethod for the Luantitation of Aicrogram
Luantities of Protein "tili8ing the Principle of Protein&7ye BindingK Anal.
Biochem. 3$!)-1&)5-.
5. anon. K+igma Aodified (o%ry Protein Assay*K +igma Procedure Do. P 5>5>.
>. anon. 1??1 KB5A Protein Assay Reagent*K Pierce catalog p. F&11.
=. anon. 1??1 KBio&Rad Protein Assay*K Bio&Rad catalog p. >2.
1. anon. 1??1 K5oomassie Plus Protein Assay ReagentK Pierce catalog p. F&)).
?. 5a$i$* I. and Polacheck* B. 1?1- KProtein assay for dilute solutions.K Methods in
Enzymology* /%4!311&3)1.
12. anon. 1??1 KFast Protein Assay*K Pierce catalog F&)>.