1. Comparison of Different Photometric Methods to Measure Protein Concentrations
Marika Raitio, Jorma Lampinen, Arto Perälä and Michelle Smits
Thermo Fisher Scientific, Vantaa, Finland
Overview
Purpose The purpose of this paper is to evaluate three commonly used
Methods
The same BSA & BGG standard & sample series were pipetted to normal and
Results
Direct protein 280 nmp p p p p y
photometric protein concentration measurement methods on different
sample volumes.
Methods In this study the direct 280 nm and two of the colorimetric
methods; BCA and Thermo Scientific Pierce 660nm Protein, are compared
with two separate proteins. The plate format from 96 to low volume 384
p p p
small volume UV 96 and 384 plates and measured according to assay
instructions.
Direct protein 280 nm
BSA and BGG sample series (8 dilutions each in 2 parallels in plate) were
pipetted to normal & low volume 96 & 384 well plates to find out the
In the table below the pathlength corrected absorbances of each sampe are
compared the the calculated theoreticl absorbance.
well plates are used.
Results The results show that the Thermo Scientific reagents and
microplate instrumentation can reliably and simply be used to determine
the protein concentration on several different methods and sample formats
from a few micrograms to several thousands of micrograms per milliliter.
It i l h th t h th l l il bl i li it d it i
characteristics in different plate formats.
Sample volumes and the corresponding approximate pathlengths.
Normal 96 Low volume 96 Normal 384 Low volume 384
Assay volume (µl) 120 80 40 20
It is also shown, that when the sample volume available is limited, it is
possible to quantify the proteins on a microplate even from volumes down
to 20 µl or less without compromising the performance.
Introduction
Direct protein 280 nm
The 384 well plates were centrifuged for 1 minute at 1000 rpm.
The absorbances were measured with Multiskan GO at 280 nm using
pathlength correction option of the SkanIt software.
Pathlength (cm) 0.3 0.5 0.3 0.4
The theoretical detection range based on the instrument precision and linear range
specifications, 0.003 and 2.5 Abs, respectively, are: BSA 14 µg/ml-3700 µg/ml and
and BGG 7 µg/ml – 1800 µg/ml.
The results gained correlate well with those values. The difference between the
plate formats can clearly be seen on the lower end of the concentration area. The
Direct protein 280 nm
The direct method is based on the absorption of light in the amino acids
containing aromatic side chains. Those are the primary reason for the
absorbance peak at 280 nm.
The protein concentration can be calculated from the formula: A = ε c L = ε
c, C=A/ ε, when L = 1 cm.
As always in photometric assays the pathlength is an important factor in
Extinction coefficients used were 0.66 for BSA and 1.36 for BGG.
low volume 96 well plate gives the best results because of the longest pathlength.
Even 20 µl or lower can be used as the sample volume on a low volume 384 well
plate.
BCA and Pierce 660 protein assay
As always in photometric assays, the pathlength is an important factor in
the assay performance.
For the direct method, the theoretical linear assay range can be calculated
from the instrument parameters, i.e instrument precision/ ε – instrument
linear range / ε. However this value is theoretical, and in normal assay
environment it can’t never be reached.
The standard curves of both dye based assays are shown on the figures below for
both BSA and BGG standards. As can be seen from the data, it is really important
to choose a protein as close as possible to the sample protein.
660 protein assay
The Pierce 660 nm Protein Assay is based on a proprietary dye-metal
complex that binds to proteins in an acidic solution. Upon binding, the
reddish dye-metal complex turns green, resulting in an absorbance shift
measurable at 660 nm.
The Pierce® 660 nm Protein Assay gives even more linear response than
Figure 1. Pathlength correction in SkanIt software for Multiskan GO
the Bradford method, and it is compatible with high concentrations of most
detergents and reducing agents. The assay is also fast to perform – the
incubation takes only 5 minutes.
BCA
In the BCA protein assay, proteins reduce Cu+2 to Cu+1 in an alkaline
1
BCA Protein assay
BSA and BGG standard and sample series (8 dilutions each in 2 parallels) were
measured in normal & low volume 96 & 384 well plates. The samples were
diluted 1:8 with BCA Working reagent.
Normal 96 Low volume 96 Normal 384 Low volume 384
As a summary the concentrations gained on each method are collected to the
tables below. As can be seen from the results, the difference for this type of pure
samples is really small. With a good quality photometer all of the assays and plate
formats are usable for most of the further applications.
medium. The BCA (bicinchoninic) molecule chelates the Cu+1 ion, which
results in a purple product with a strong absorbance at 562nm.
As a method the BCA is less prone to interference than the Lowry
procedure and is more sensitive.
Materials
The 384 well plates were centrifuged for 1 minute at 1000 rpm before and after
reagent addition. The plates were mixed for 20 s and incubated for 30 min at
37°C in iEMS Incubator/Shaker and measurement at 562 nm (Multiskan GO).
BSA Direct 280 nm BCA 660
Theoretical
Concentration
(µg/ml)
Normal
96
Low vol
96
Normal
384
Low vol
384
Normal
96
Low vol
96
Normal
384
Low vol
384
Normal
96
Low vol
96
Normal
384
Low vol
384
138 136 132 146 140 177 142 150 129 144 144 173 145
275 263 269 272 277 334 291 304 388 302 263 301 294
550 528 529 546 528 625 570 630 557 576 511 520 591
1100 1051 1044 1080 1060 1018 1113 1155 1170 1100 1010 1034 1235
Assay volume (µl) 120 80 40 20
Materials
Reagents
Thermo ScientificTM Pierce Bovine Gamma Globulin Standard (BGG), 2
mg/ml (Cat no 23212)
Thermo ScientificTM Pierce Albumin Standard (BSA), 2 mg/ml (Cat no
23210)
The reported assay linear range is 125-2,000μg/mL on a microplate, when the
sample volume is 10 µl.
Pierce 660 nm protein assay
BSA and BGG standard and sample series (8 dilutions each in 2 parallels) were
measured in normal & low volume 96 & 384 well plates The samples diluted
BGG Direct 280 nm BCA 660
Theoretical
Concentration
(µg/ml)
Normal
96
Low vol
96
Normal
384
Low vol
384
Normal
96
Low vol
96
Normal
384
Low vol
384
Normal
96
Low vol
96
Normal
384
Low vol
384
156 120 113 117 110 145 145 201 188 143 123 144 168
313 260 253 256 252 305 317 389 355 314 274 278 292
625 544 549 550 542 596 680 639 622 649 585 655 595
1250 1153 1152 1174 1135 1264 1374 1232 1236 1239 1250 1168 1194
Conclusion
The method to be used should always be chosen based on several
factors; performance need, sample type and volume
It is important to use good quality instrumentation and plates and the
)
Thermo Scientific PierceTM BCATM Protein assay kit (Cat no 23227)
Thermo Scientific PierceTM 660 Protein assay (Cat no 22660)
Plates
Greiner Bio-one UV-Star 96 well plate (Cat no 655801) (Normal 96)
Greiner Bio-one UV-Star 96 well half area plate (Cat no 675801) (Low
measured in normal & low volume 96 & 384 well plates. The samples diluted
1:16 with 660 nm Protein Reagent.
1250 1153 1152 1174 1135 1264 1374 1232 1236 1239 1250 1168 1194
Normal 96 Low volume 96 Normal 384 Low volume 384
Assay volume (µl) 160 96 58 24
It is important to use good quality instrumentation and plates, and the
standards to be used with the dye-based material need to be similar to
the unknown protein
Especially in the direct method the sample volume ( pathlength) is
critical
volume 96)
Greiner Bio-one UV-Star 384 well plate (Cat no 781801) (Normal 384)
Greiner Bio-one µClear 384 well plate (Cat no 788876) (Low volume 384)
Instruments
Thermo Scientific Multiskan GO microplate spectrophotometer (Cat no
51119300)
Mixing (96 well plates) or centrifugation (384 well plates): 1000 rpm, 1 min
before & after reagent addition.
The plates were incubated for 5 min at RT and measured at 660 nm (Multiskan
GO)
The reported assay linear range is 50-2,000μg/mL on a microplate.
51119300)
Thermo Scientific iEMS Incubator/Shaker (Cat no 5112250) In the both of the reagent based assays a set of standards in the concentration
range of 25–2000 μg/ml was used to create the standard curve. The buffer
solution was used as a blank sample.
In this test the sample volumes were in some cases below the recommended
volumes, and therefore the working ranges specified can’t exactly be expected.
.