ANALYTI CAL BI OCHEMI STRY 236, 302308 (1996)

ARTI CLE NO. 0171

Linear izat ion of t he Br adfor d Pr ot ein Assay Incr eases It s
Sensit ivit y: Theor et ical and Exper iment al St udies
Tsaffri r Zor and Zvi Sel i nger
1
Department of Biological Chemistry and theKuhneMinerva Center for Studies of Visual Transduction,
TheHebrew University of J erusalem, J erusalem91904, I srael
Recei ved November 20, 1995
str ucti on of the cal i br ati on gr aph. Thi s nonl i near i ty
Determination of microgramquantitiesof proteinin
pr esents a ser i ous pr obl em outsi de thi s nar r ow
theBradford Coomassiebrilliant blueassay is accom-
r ange of pr otei n concentr ati ons and when mi cr ogr am
plished by measurement of absorbance at 590 nm.
amounts of pr otei n ar e not avai l abl e. Over 100 wor ks
However, an intrinsic nonlinearity compromises the
have attempted to i mpr ove the Br adfor d assay (for
sensitivity and accuracy of this method. It is shown
r evi ew, see (2)). Some have addr essed the nonl i near -
that under standard assay conditions, the ratio of the
i ty pr obl em (35), but al l offer ed onl y a par ti al sol u-
absorbances,590nmover 450nm,isstrictlylinear with
ti on.
protein concentration. This simple procedure in-
Thr ee char ge for ms of the Coomassi e br i l l i ant bl ue
creases the accuracy and improves the sensitivity of
dye ar e pr esent i n equi l i br i um at the usual aci di c
theassay about 10-fold, permittingquantitation down
pH of the assay. The r ed, bl ue, and gr een for ms have
to 50 ng of bovine serum albumin. Furthermore, pro-
absor bance maxi ma at 470, 590, and 650 nm, r espec-
tein assay in presenceof up to35-foldweight excessof
ti vel y (6). The bl ue i s the for m that bi nds the pr otei n,
sodiumdodecyl sulfate(detergent) over bovineserum
for mi ng a compl ex that i ntensel y absor bs l i ght at 594
albumin (protein) can beperformed. A linear equation
nm (7, 8). Ther efor e one may moni tor the decr ease i n
that perfectly ts the experimental data is provided
concentr ati on of the fr ee bl ue for m i n the pr esence
onthebasisof massactionandBeerslaw. 1996Academic
of pr otei n as r eected by the decr ease i n absor bance
Press, Inc.
at 450 nm that i s pr opor ti onal mai nl y to the concen-
tr ati on of the r ed dye for m. Fol l owi ng thi s pr ocedur e,
we have found that i n contr ast to Br adfor ds pr oposal
The Coomassi e br i l l i ant bl ue pr otei n assay, known
the decr easi ng backgr ound coul d not ful l y account
as the Br adfor d assay (1), i s wi del y used because of
for the nonl i near i ty. The decr ease i n dye concentr a-
i ts ease of per for mance, r api di ty, r el ati ve sensi ti vi ty,
ti on, however , does pr oduce another di stor ti on of the
and speci ci ty for pr otei ns. As Br adfor d her sel f ob-
l i near r esponse because bi ndi ng to the pr otei n i s i n
ser ved, however , Ther e i s a sl i ght nonl i near i ty i n
equi l i br i um (9); thus compl ex for mati on i s depen-
the r esponse patter n. The sour ce of the nonl i near i ty
dent not onl y on pr otei n concentr ati on but al so on
i s i n the r eagent i tsel f si nce ther e i s an over l ap i n
dye concentr ati on. Thi s noti on had been pr evi ousl y
the spectr um of the two di ffer ent col or for ms of the
descr i bed by Chi al and Spl i ttger ber (7).
dye. The backgr ound val ue for the r eagent i s conti n-
The pr esent study shows that when these two i n-
ual l y decr easi ng as mor e dye i s bound to pr otei n
ter fer ences ar e taken i nto account i n Beer s l aw, an
(1). Over a br oad r ange of pr otei n concentr ati ons the
equati on can be wr i tten that descr i bes a l i near r el a-
degr ee of cur vatur e i s qui te l ar ge; ther efor e, onl y a
ti onshi p between pr otei n concentr ati on and the r ati o
nar r ow r ange of r el ati vel y hi gh pr otei n concentr a-
of absor bances, 590 nm over 450 nm. Thi s equati on
ti ons, 2 10 mg/ml BSA,
2
i s used for assay and con-
was exper i mental l y tested and found to yi el d a ful l -
scal e l i near cal i br ati on l i ne over the enti r e r ange
1
To whom correspondence shoul d be addressed at Department of
Bi ol ogi cal Chemi stry, The Hebrew Uni versi ty of Jerusal em, Jerusa-
studi ed. Fur ther mor e, the sensi ti vi ty of the Br adfor d
l em 91904, I srael . Fax: 972-2-6527427.
assay i s i ncr eased by appr oxi matel y one or der of
2
Abbrevi ati ons used: BSA, bovi ne serum al bumi n; CBBG, Coomas-
magni tude, maki ng i t possi bl e to deter mi ne as l i ttl e
si e bri l l i ant bl ue G-250; DDW, doubl e-di sti l l ed water; SDS, sodi um
dodecyl sul fate. as 50 ng BSA i n the 0.25-ml mi cr opl ate assay. The
302 0003-2697/96 $18.00
Copyri ght 1996 by Academi c Press, I nc.
Al l ri ghts of reproducti on i n any form reserved.
/ 6m14$$9509 03-27-96 10:24:34 aba AP-Anal Bio
LI NEARI ZATI ON OF BRADFORD PROTEI N ASSAY 303
i mpr oved sensi ti vi ty r esul ts al so i n r educed i nter fer -
ence by deter gents.
MATERIALS AND METHODS
Reagents
Coomassi e bri l l i ant bl ue G-250 (CBBG) was obtai ned
from Merck. Crystal l i zed bovi ne serum al bumi n was
purchased from Schwarz/Mann (Spri ng Val l ey, NY). a-
Chymotrypsi n (C-3142) was obtai ned from Si gma. Al l
reagents were of the hi ghest avai l abl e grade. Dei on-
i zed, doubl e-di sti l l ed water (DDW) was used. Protei ns
were di ssol ved i n DDW.
DyeReagent
Dye reagent was prepared accordi ng to Bradford (1).
Coomassi e bri l l i ant bl ue G-250 (100 mg) was di ssol ved
i n 50 ml 95% ethanol . A vol ume of 100 ml phosphori c
FIG.1. Spectra of the dyeprotei n compl ex (A) and the dye (CBBG)
aci d (85% w/v) was added and the sol uti on was di l uted al one (B) were obtai ned by addi ti on of 0.8 ml dye reagent to 0.2 ml
of 5 mg/ml BSA (A) or to 0.2 ml DDW (B). The l ength of the opti cal
to 1 l i ter wi th DDW and i mmedi atel y l tered twi ce.
path i s 0.1 cm i n A and 1 cm i n B. The arrows poi nt at the absorbance
The dye reagent was stored at 4C, protected from l i ght.
maxi ma of 594 and 466 nm.
Onl y when speci cal l y menti oned i n the text or gure
l egend, dye reagent from Bi o-Rad was used.
wi th the protei n, and the green form that peaks at 650
Protein Determination
nm but absorbs si gni cantl y at 590 nm, the l
max
of the
dyeprotei n compl ex (6). Si nce the dyeprotei n com-
1-ml assay. Protei n determi nati on was performed
pl ex does not absorb at 466 nm, i t was possi bl e to test
wi th a sl i ght modi cati on to the ori gi nal assay (1). A
Bradfords suggesti on (1) that the nonl i neari ty of the
vol ume of 0.8 ml dye reagent was added to dupl i cate
protei n cal i brati on curve i s due to a decrease i n the
0.2-ml protei n sampl es i n di sposabl e pl asti c cuvettes,
background val ue of the reagent as more dye bi nds to
the tube content was thoroughl y mi xed, and ab-
the protei n when protei n concentrati ons are i ncreased.
sorbance was measured after 560 mi n agai nst DDW
Beers l aw states that A ecl, where A i s the ab-
as bl ank. Al l protei n concentrati ons shown correspond
sorbance at any wavel ength, e i s the mol ar absorpti on
to the nal assay vol ume.
coefci ent at the same wavel ength, c i s the mol ar con-
0.25 ml microplate assay. The procedure was per-
centrati on of the chromophore, and l i s l ength of the
formed as descri bed for the 1-ml assay, reduci ng al l
opti cal path i n centi meters. Usi ng Beers l aw, the ex-
vol umes to
1
4
.
pressi on for the concentrati on-corrected bl ank i s the
noncorrected bl ank (A
594
wi thout protei n) mul ti pl i ed by
Equipment
the rati o of absorbances at 466 nm wi th and wi thout
protei n. We found that subtracti on of a corrected bl ank
A Vari an Cary 1E UV/VI S spectrophotometer was
from A
594
obtai ned i n the presence of 020 mg/ml BSA
used for the 1-ml assay. Mi cropl ate autoreader EL309
does not ful l y l i neari ze the response curve, i ndi cati ng
of BI O-TEK I nstruments was used for the 0.25-ml
an addi ti onal cause for the nonl i neari ty (resul ts not
assay.
shown).
Si nce dyeprotei n compl ex formati on i s not l i mi ted
RESULTS AND DISCUSSION
by the amount of protei n, yet the dye i s not i n a l arge
Theoretical Considerations
excess (10), we concl uded that the formati on of the dye
protei n compl ex wi l l not be proporti onal to protei n con-
The spectrum (Fi g. 1A) of the dyeprotei n compl ex
centrati on and wi l l be dependent on dye concentrati on
(l
max
594 nm) was obtai ned by addi ti on of the dye
and on the coefci ent of the equi l i bri um reacti on
reagent to a l arge mol ar excess of protei n. Under thi s
condi ti on, there are practi cal l y no free dye mol ecul es,
as demonstrated by the compl ete di sappearance of the
D / P }
K
DP [1]
red dye form that absorbs at l
max
466 nm (Fi g. 1B).
The free dye exi sts as the aci d base equi l i bri um of the i n whi ch P i s the protei n and D i s the dye. The equi l i b-
ri um constant i s dened as red (l
max
466 nm) form, the bl ue form that reacts
/ 6m14$$9509 03-27-96 10:24:34 aba AP-Anal Bio
ZOR AND SELI NGER 304
compati bl e wi th the mi cropl ate autoreader i nstrument.
K
[DP]
[nP] 1 [D] 1 f
b
[2]
I t shoul d be poi nted out that e
D
, at both 590 and 450
nm, i s a wei ghted average of the mol ar absorpti on coef-
ci ents of the three dye forms. The addi ti on of protei n
where f
b
, the fracti on of the reacti ve bl ue dye form,
does not change the combi ned e
D
si nce the fracti on of
equal s 0.053 under assay condi ti ons (6) and n i s the
each dye form depends onl y on pH and not on total
number of bi ndi ng si tes on a protei n mol ecul e, bei ng
concentrati on or consumpti on of one dye form. The sub-
pri mari l y argi ni ne resi dues (8). For si mpl i ci ty the bi nd-
sti tuti on of Eq. [7] i nto Eq. [6] resul ts i n
i ng si tes are assumed to be homogeneous and noni nter-
acti ng, al though bi ndi ng has some uncl ear dependence
on macromol ecul ar structure (8). Substi tuti ng the con-
A
590

e
DP
590
1 l

e
D
450
1 l
K 1 f
b
1 A
450
/ 1

servati on equati on of protei n bi ndi ng si tes, [nP]

t
[nP]
/ [DP], where t i ndi cates total , i nto Eq. [2] and rear-
rangement yi el ds
1 [nP]
t
/
e
D
590
1 l
e
D
450
1 l
1 A
450
[8]
[DP]
K 1 f
b
1 [D]
1 / K 1 f
b
1 [D]
1 [nP]
t
. [3]
whi ch i s rearranged to become
Further rearrangement gi ves the fol l owi ng expres-
si on for the compl ex concentrati on:
A
590
A
450

e
DP
590
1 l

e
D
450
1 l
K 1 f
b
/ A
450

1 [nP]
t
/
e
D
590
e
D
450
. [9]
[DP]
1

1
K 1 f
b
1 [D]
/ 1

1 [nP]
t
. [4]
To reach a l i near equati on, i n whi ch protei n concen-
trati on i s the X vari abl e, the fol l owi ng assumpti on i s
The absorbance at 590 nm, composed of the i ndepen-
made
dent absorbances of both the dyeprotei n compl ex and
the free dye, i s gi ven by
e
D
450
1 l
K 1 f
b
A
450
. [10]
A
590
e
DP
590
1 l 1 [DP] / e
D
590
1 l 1 [D], [5]
Substi tuti on of Eqs. [2] and [7] i nto Expressi on [10]
where the superscri pt of e denes the compound and
and rearrangement gi ves
the subscri pt refers to the wavel ength.
Substi tuti on of Eq. [4] i nto Eq. [5] l eads to
[nP]
[DP]
1. [11]
A
590

e
DP
590
1 l

1
K 1 f
b
1 [D]
/ 1

The cri ti cal assumpti on gi ven by Expressi ons [10]

and [11] was veri ed by the el egant experi ments of
Spl i ttgerber and col l eagues (6, 9). Accordi ng to these
1 [nP]
t
/ e
D
590
1 l 1 [D]. [6]
experi ments, the rati o [nP] /[DP] equal s approxi matel y
4 when consi deri ng onl y hi gh-afni ty bi ndi ng si tes. I n
Accordi ng to Eq. [6], both components of A
590
depend
thi s case, the val ue of the expressi on precedi ng [nP]
t
upon free dye concentrati on. Thus the correcti on for
i n Eq. [9] changes by l ess than 5% over the protei n
the absorbance of the free dye al one wi l l not l ead to
concentrati ons range assayed. Furthermore, when con-
l i near dependence between A
590
and protei n concentra-
si deri ng l ow-afni ty bi ndi ng si tes, the rati o [nP] /[DP]
ti on.
i s i ndeed far l arger than 1 as assumed i n Expressi on
Because the dyeprotei n compl ex has no contri bu-
[11] and the val ue of the expressi on precedi ng [nP]
t
ti on to the absorbance at 450 nm, [D] can be cal cul ated
i n Eq. [9] i s practi cal l y constant. Si nce under assay
as fol l ows:
condi ti ons a substanti al part of the absorbance i s con-
tri buted by l ow-afni ty bi ndi ng si tes (9), i t can be con-
A
450
e
D
450
1 l 1 [D]. [7]
cl uded that the assumpti on gi ven by Expressi ons [10]
and [11] i s ful l y justi ed.
Therefore, when assumi ng rel ati onshi p [10], Expres- Thi s wavel ength, l i ke 590 nm, was chosen rather
than the l
max
(466 and 594 nm) to make the assay si on [9] becomes
/ 6m14$$9509 03-27-96 10:24:34 aba AP-Anal Bio
LI NEARI ZATI ON OF BRADFORD PROTEI N ASSAY 305
A
590
A
450

e
DP
590
1 K 1 f
b
1 n
e
D
450
1 [P]
t
/
e
D
590
e
D
450
. [12]
Equati on [12] predi cts a l i near rel ati onshi p between
the rati o A
590
/A
450
and total protei n concentrati on, [P]
t
,
when the bi ndi ng i s far from saturati on (Expressi on [11]).
As expl ai ned above, for si mpl i ci ty the bi ndi ng si tes
were assumed to be homogeneous and noni nteracti ng.
Under assay condi ti ons, however, bi ndi ng to a hetero-
geneous popul ati on of bi ndi ng si tes occurs (9). Yet, we
found that the same form of l i near equati on (Eq. [12])
i s obtai ned. The sl ope of thi s equati on i ncl udes the pa-
rameters of al l di fferent bi ndi ng si tes: equi l i bri um con-
stants (K) and number of bi ndi ng si tes on a protei n
mol ecul e (n).
FIG. 3. Pl ots for the free CBBG dye at 450 nm (l) and at 590 nm
() under the usual assay condi ti ons.
Experimental Support of theTheory
The fol l owi ng observati ons are consi stent wi th the shows a strai ght l i ne obtai ned upon pl otti ng A
590
/A
450
l i near Eq. [12]: as a functi on of BSA concentrati on.
(a) Whi l e the regul ar Bradford cal i brati on graph (b) The theoreti cal equati on predi cts that the l i near
(Fi g. 2A) markedl y devi ates from l i neari ty, Fi g. 2B curve wi l l i ntercept the Y axi s at a val ue that equal s
the rati o of the dyes mol ar absorpti on coefci ent at
590 nm over that at 450 nm. These constants were
determi ned and found to be 5200 and 11,300 M
01
cm
01
,
respecti vel y (Fi g. 3). The rati o of the mol ar absorpti on
coefci ents i s 0.46 { 0.01. Thi s val ue i s i n excel l ent
agreement wi th the experi mental data of the cal i bra-
ti on graph whi ch i s 0.48 { 0.02 (average of 15 i ndepen-
dent determi nati ons, cf. Fi gs. 2B, 4, 5, and 6)
Calibration Graph
The regul ar Bradford cal i brati on graph (Fi g. 2A)
shows di sti nct curvature i n the range of 020 mg/ml
FIG. 4. Li neari zati on of the Bradford cal i brati on graph i n the 0.25-
ml mi cropl ate assay. The strai ght l i ne was produced by the val ues
deri ved of 7 poi nts usi ng 04 mg BSA. Onl y the range of 0100 ng FIG. 2. Cal i brati on graphs of 020 mg/ml BSA. (A) The conven-
ti onal Bradford cal i brati on graph. The l i near regressi on l i ne was BSA i s shown for the demonstrati on of sensi ti vi ty. The sl ope of the
graph i s smal l er than that of Fi g. 2B by approxi matel y 250 due to cal cul ated for the narrow range of 210 mg/ml BSA (). (B) Li near-
i zed Bradford cal i brati on graph. The same set of sampl es was used the 4-fol d smal l er protei n quanti ti es and the 3 orders of magni tude
di fference i n X-axi s uni ts. for both pl ots.
/ 6m14$$9509 03-27-96 10:24:34 aba AP-Anal Bio
ZOR AND SELI NGER 306
The procedure descri bed can be appl i ed to any stan-
dard protei n, as shown i n Fi g. 5. Li near curves were
obtai ned for both a-chymotrypsi n and BSA, whi l e the
response of BSA i s 2.8-fol d hi gher than the response of
a-chymotrypsi n, a val ue that i s i n very good agreement
wi th previ ous reports (11, 12). For both protei ns the
regul ar cal i brati on graph was nonl i near (resul ts not
shown). I t shoul d be kept i n mi nd that protei n determi -
nati on i s al ways rel ati ve to the standard and not abso-
l ute. BSA i s a commonl y used standard i n the Bradford
assay because of i ts hi gh col or yi el d and avai l abi l i ty
whi l e other standards may more cl osel y resembl e the
protei ns under determi nati on (Fi g. 5 and Ref. (13)).
FIG. 5. Li neari zati on of the Bradford cal i brati on graph usi ng a-
Determination of Unknown Protein Samples
chymotrypsi n (l) and BSA (). The l i near equati ons are Y 0.053X
/ 0.474 wi th R
2
0.998 for a-chymotrypsi n and Y 0.146X / 0.457 Al though Fi g. 4 shows that 0.2 mg/ml BSA can be
wi th R
2
0.999 for BSA.
accuratel y determi ned, the l i mi ts of detecti on of an un-
known protei n sampl e must al so be establ i shed. To thi s
end, rabbi t serum was di l uted to gi ve 3110 nl per
BSA. However, i n a curved graph, a semi l i near rel a-
ti onshi p can be seen over a narrow range of poi nts. The
cl ose to l i near range of the Bradford cal i brati on graph
i s consi dered to be 210 mg/ml BSA si nce smal l er pro-
tei n concentrati ons are characteri zed by a smal l si gnal
to noi se rati o and therefore cannot be accuratel y deter-
mi ned from a nonl i near graph. I t i s i mportant to note
that the l i near regressi on i n Fi g. 2A was cal cul ated
usi ng onl y 5 poi nts (shown as squares) wi thi n the l i n-
ear range. Fi gure 2B shows that a l i near correl ati on
exi sts between A
590
/A
450
and BSA concentrati on i n the
range of 020 mg/ml , for the same set of sampl es used
i n pl otti ng the nonl i near Fi g. 2A. Stati sti cal anal ysi s
gi ves standard devi ati ons of 1.2 and 0.9% of error i n
the narrow range of 210 mg/ml BSA, for the regul ar
and corrected cal i brati on curves, respecti vel y. The cor-
rel ati on parameter, R
2
, i s i ncreased from 0.976 i n Fi g.
2A to 0.999 i n Fi g. 2B. The di fference i s even more
obvi ous i n the ful l -scal e graphs, where the standard
devi ati on i s decreased from 3.9% error for the regul ar
cal i brati on curve to 1.2%error for the corrected cal i bra-
ti on curve. The stati sti cal anal ysi s proves the i mprove-
ment i n l i neari ty over the standard concentrati ons
range and demonstrates the l i near rel ati onshi p be-
tween A
590
/A
450
rati o and protei n concentrati on over
the enti re range, 020 mg/ml .
I n order to determi ne the l i mi ts of sensi ti vi ty, an
extended range of protei n concentrati ons was tested. A
quanti ty of 0.2 mg BSA can be accuratel y determi ned
i n the 1-ml assay whi l e Fi g. 4 shows that an amount
as l ow as 50 ng BSA i s accuratel y determi ned i n the FIG. 6. Cal i brati on graphs i n the absence () or presence (l) of
0.002%SDS. A vol ume of 0.8 ml dye reagent from Bi o-Rad was added
0.25-ml mi cropl ate assay. The l i near rel ati onshi p ex-
to 0.2 ml protei n sampl e wi thout () or wi th (l) SDS. (A) Conven-
i sts up to 20 mg BSA i n the 1-ml assay and up to 4 mg
ti onal Bradford cal i brati on graph. (B) Li neari zed Bradford cal i bra-
BSA i n the mi cropl ate assay. I t can be concl uded that
ti on graph. The same set of sampl es was used for both pl ots. The
the procedure presented here produces a ful l -scal e l i n-
l i near equati ons are Y 0.205X / 0.457 wi th R
2
1.000 () and Y
0.070X / 0.452 wi th R
2
0.999 (l). ear graph.
/ 6m14$$9509 03-27-96 10:24:34 aba AP-Anal Bio
LI NEARI ZATI ON OF BRADFORD PROTEI N ASSAY 307
TABLE 1
Determi nati on of Serum Protei n
a
Serum protei n
c
SD percentage
f
mg/ml i n the serum
e
No. Serum (ml ) A
590
/A
450
b
mg i n assay
d
By one poi nt By sl ope
e
By one poi nt By sl ope
e
1 0 0.462
2 0.003 0.480 0.17 51.0 53.0 7.3 4.8
3 0.010 0.530 0.57 61.5 62.8 5.7 7.3
4 0.014 0.547 0.80 54.9 57.3 2.4 0.6
5 0.070 0.898 3.98 57.0 57.1 0.1 0.2
6 0.110 1.144 6.26 56.8 56.9 0.1 0
a
1-ml assay.
b
Average of dupl i cate sampl es.
c
Equati on of the BSA standard curve: Y 0.109X / 0.463.
d
The concentrati on used for the cal cul ati on i s 56.9 mg/ml , determi ned by the rati o of the sl opes.
e
The sl ope was determi ned by the curve of sampl es 1N (N 26).
f
Standard devi ati on of the concentrati on from 56.9 mg/ml .
assay and these were determi ned i n paral l el to BSA i n of detergents from the sampl e (15). The i nterference
becomes evi dent when nal SDS concentrati on exceeds a standard cal i brati on curve. The l i near equati ons of
BSA and serum are Y 0.109X / 0.463 and Y 6.20X 0.002% (15). Di l uti ng the sol ubi l i zed protei n sampl e i n
order to reach noni nterferi ng detergent concentrati on / 0.463, respecti vel y, wi th correl ati on parameters R
2
of 0.996 and 0.999, respecti vel y. The Y val ues are A
590
/ frequentl y resul ts i n protei n content that i s bel ow the
threshol d of detecti on. Fi gures 6A and 6B show the A
450
and the X val ues are mi crograms of BSA or mi cro-
l i ters of serum. I n general , the most accurate method regul ar and corrected cal i brati on graphs, respecti vel y,
i n presence of 0.002%SDS. A l i near rel ati onshi p exi sts to determi ne protei n concentrati on i n an unknown
sampl e i s to di vi de the sl ope of several sampl e di l uti ons between the rati o A
590
/A
450
and protei n quanti ty i n
both the absence and presence of 0.002% SDS. I t i s by the sl ope of the cal i brati on curve. The product repre-
senti ng concentrati on uni ts i s the protei n concentra- therefore possi bl e to determi ne as l i ttl e as 0.3 mg/ml
of protei n sol ubi l i zed by 1% SDS. The response to pro- ti on of the unknown sampl e before di l uti on. Tabl e 1
shows the resul ts of these cal cul ati ons. The error i s tei n i s reduced by the detergent, whi l e the background
i s constant. However, the sl ope of the A
590
/A
450
curve i ncreasi ng gradual l y from 0.1% for 6.26 mg up to 7.3%
for the sampl e contai ni ng onl y 0.17 mg i n the 1-ml i n presence of 0.002% SDS i s about equal to the sl ope
of the A
590
curve i n the absence of detergents (compare assay. The error i s markedl y reduced when the concen-
trati on i s cal cul ated by the sl ope of the unknown sam- Fi gs. 2A, 2B, and 6B). Hi gher SDS concentrati ons con-
si derabl y reduce the response to protei n and i ncrease pl e curve made of 26 poi nts, i ncl udi ng the bl ank. Ta-
bl e 1 shows that i t sufces to determi ne the sl ope the background. Therefore, nal SDS concentrati on
that exceeds 0.002% does not al l ow protei n determi na- between one poi nt of protei n concentrati on and the
bl ank val ue at zero protei n concentrati on, to si gni - ti on. These ndi ngs and Eq. [12] are consi stent wi th
previ ous suggesti on (16) that a hi gh SDS concentrati on cantl y i mprove the accuracy (Tabl e 1, l i ne 2). Usual l y,
above 1 mg protei n, cal cul ati on by one poi nt i s accurate i nterferes wi th the Bradford assay by stabi l i zi ng the
green dye form whi l e a l ow SDS concentrati on i nter- enough and a sl ope i s not needed. Si mi l ar resul ts were
obtai ned i n the mi cropl ate assay, testi ng 421560 ng feres by competi ng wi th the dye on bi ndi ng to the pro-
tei n. As shown i n Fi g. 6B, the l atter i nterference can protei n of rabbi t serum, al though the errors were some-
what hi gher, due to i nstrumental opti cal l i mi tati ons. be el i mi nated by measurement of A
590
/A
450
i n the pres-
ence of the detergent for both the standard and the
unknown sampl e.
I nterferenceby Detergents
Detergents are known to i nterfere wi th the Bradford
CONCLUSIONS
assay (1). Protei n determi nati on of sol ubi l i zed protei ns
by Bradford assay can be carri ed out when gl ucopyra- The purpose of the present study was to eval uate the
reasons for the nonl i neari ty of the Bradford assay i n nosi de detergents are used (14) or fol l owi ng excl usi on
/ 6m14$$9509 03-27-96 10:24:34 aba AP-Anal Bio
ZOR AND SELI NGER 308
order to devel op a procedure that woul d yi el d a l i near consi derati ons. We hope that the i mproved accuracy
and sensi ti vi ty and the parti al el i mi nati on of i nterfer- rel ati onshi p between absorbance and protei n concen-
ence by detergents wi l l promote a more wi despread use trati on. We have found that the nonl i neari ty i s due to
of the assay. two factors; both of them ori gi nate from the fact that
the free dye concentrati on i s decreased by protei n addi -
ACKNOWLEDGMENTS
ti on. The outcome i s a conti nuous decrease i n the ab-
sorbance contri buted by the free reagent on the one
We thank Dr. M. Schramm, Dr. S. Brawn, Dr. O. Hei chal , and M.
hand and a reduced dyeprotei n compl ex formati on on Dani n for hel pful comments on the manuscri pt. The authors re-
search was supported by grants from the Nati onal I nsti tute of Heal th
the other hand. An equati on descri bi ng absorbance at
(Ey-03529) and the Uni ted States I srael Bi nati onal Sci ence Founda-
590 nm, taki ng i nto account these two factors, demon-
ti on.
strates a l i near rel ati onshi p between the rati o of ab-
sorbances at 590 nm over 450 nm and total protei n
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/ 6m14$$9509 03-27-96 10:24:34 aba AP-Anal Bio