Comparison of Different Photometric Methods to Measure Protein Concentrations

Marika Raitio, Jorma Lampinen, Arto Perl and Michelle Smits

Thermo Fisher Scientific, Vantaa, Finland

Overview Methods Results

Purpose
p The p purpose
p of this p
paper
p is to evaluate three commonly
y used The same BSA & BGG standard & sample
p series were p
pipetted
p to normal and Direct protein 280 nm
photometric protein concentration measurement methods on different small volume UV 96 and 384 plates and measured according to assay In the table below the pathlength corrected absorbances of each sampe are
sample volumes. instructions. compared the the calculated theoreticl absorbance.

Methods In this study the direct 280 nm and two of the colorimetric Direct protein 280 nm
methods; BCA and Thermo Scientific Pierce 660nm Protein, are compared BSA and BGG sample series (8 dilutions each in 2 parallels in plate) were
with two separate proteins. The plate format from 96 to low volume 384 pipetted to normal & low volume 96 & 384 well plates to find out the
well plates are used. characteristics in different plate formats.

Results The results show that the Thermo Scientific reagents and Sample volumes and the corresponding approximate pathlengths.
microplate instrumentation can reliably and simply be used to determine
the protein concentration on several different methods and sample formats Normal96 Lowvolume 96 Normal384 Lowvolume384
from a few micrograms to several thousands of micrograms per milliliter. Assayvolume(l) 120 80 40 20
It is
i also
l shown,
h th t when
that h th sample
the l volume
l available
il bl is
i limited,
li it d it is
i Pathlength(cm) 0.3 0.5 0.3 0.4
possible to quantify the proteins on a microplate even from volumes down The theoretical detection range based on the instrument precision and linear range
to 20 l or less without compromising the performance. specifications, 0.003 and 2.5 Abs, respectively, are: BSA 14 g/ml-3700 g/ml and
The 384 well plates were centrifuged for 1 minute at 1000 rpm. and BGG 7 g/ml 1800 g/ml.
The absorbances were measured with Multiskan GO at 280 nm using The results gained correlate well with those values. The difference between the
Introduction pathlength correction option of the SkanIt software. plate formats can clearly be seen on the lower end of the concentration area. The
Direct protein 280 nm low volume 96 well plate gives the best results because of the longest pathlength.
The direct method is based on the absorption of light in the amino acids Extinction coefficients used were 0.66 for BSA and 1.36 for BGG. Even 20 l or lower can be used as the sample volume on a low volume 384 well
containing aromatic side chains. Those are the primary reason for the plate.
absorbance peak at 280 nm.
The protein concentration can be calculated from the formula: A = c L =
c, C=A/ , when L = 1 cm. BCA and Pierce 660 protein assay
As always in photometric assays,
assays the pathlength is an important factor in
the assay performance. The standard curves of both dye based assays are shown on the figures below for
For the direct method, the theoretical linear assay range can be calculated both BSA and BGG standards. As can be seen from the data, it is really important
from the instrument parameters, i.e instrument precision/ instrument to choose a protein as close as possible to the sample protein.
linear range / . However this value is theoretical, and in normal assay
environment it cant never be reached.

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 Figure 1. Pathlength correction in SkanIt software for Multiskan GO
measurable at 660 nm.
The Pierce 660 nm Protein Assay gives even more linear response than
the Bradford method, and it is compatible with high concentrations of most BCA Protein assay
detergents and reducing agents. The assay is also fast to perform the BSA and BGG standard and sample series (8 dilutions each in 2 parallels) were
incubation takes only 5 minutes. measured in normal & low volume 96 & 384 well plates. The samples were As a summary the concentrations gained on each method are collected to the
diluted 1:8 with BCA Working reagent. tables below. As can be seen from the results, the difference for this type of pure
BCA samples is really small. With a good quality photometer all of the assays and plate
In the BCA protein assay, proteins reduce Cu+2 to Cu+1 in an alkaline Normal96 Lowvolume 96 Normal384 Lowvolume384 formats are usable for most of the further applications.
medium. The BCA (bicinchoninic) molecule chelates the Cu+11 ion, which Assayvolume(l) 120 80 40 20 BSA Direct 280 nm BCA 660

results in a purple product with a strong absorbance at 562nm. Theoretical

Normal Low vol Normal Low vol Normal Low vol Normal Low vol Normal Low vol Normal Low vol
Concentration
As a method the BCA is less prone to interference than the Lowry (g/ml)
96 96 384 384 96 96 384 384 96 96 384 384

procedure and is more sensitive. The 384 well plates were centrifuged for 1 minute at 1000 rpm before and after 138 136 132 146 140 177 142 150 129 144 144 173 145

reagent addition. The plates were mixed for 20 s and incubated for 30 min at 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
37C in iEMS Incubator/Shaker and measurement at 562 nm (Multiskan GO). 1100 1051 1044 1080 1060 1018 1113 1155 1170 1100 1010 1034 1235

Materials
The reported assay linear range is 125-2,000g/mL on a microplate, when the BGG Direct 280 nm BCA 660

Reagents sample volume is 10 l. Theoretical

Normal Low vol Normal Low vol Normal Low vol Normal Low vol Normal Low vol Normal Low vol
Concentration
Thermo ScientificTM Pierce Bovine Gamma Globulin Standard (BGG), 2 (g/ml)
96 96 384 384 96 96 384 384 96 96 384 384

mg/ml (Cat no 23212) Pierce 660 nm protein assay 156 120 113 117 110 145 145 201 188 143 123 144 168

Thermo ScientificTM Pierce Albumin Standard (BSA), 2 mg/ml (Cat no BSA and BGG standard and sample series (8 dilutions each in 2 parallels) were
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
23210)) measured in normal & low volume 96 & 384 well plates.
plates The samples diluted 1250 1153 1152 1174 1135 1264 1374 1232 1236 1239 1250 1168 1194

Thermo Scientific PierceTM BCATM Protein assay kit (Cat no 23227) 1:16 with 660 nm Protein Reagent.
Thermo Scientific PierceTM 660 Protein assay (Cat no 22660)
Normal96 Lowvolume 96 Normal384 Lowvolume384
Conclusion
Plates Assayvolume(l) 160 96 58 24 The method to be used should always be chosen based on several
Greiner Bio-one UV-Star 96 well plate (Cat no 655801) (Normal 96) factors; performance need, sample type and volume
Greiner Bio-one UV-Star 96 well half area plate (Cat no 675801) (Low
volume 96) It is important to use good quality instrumentation and plates
plates, and the
Mixing (96 well plates) or centrifugation (384 well plates): 1000 rpm, 1 min standards to be used with the dye-based material need to be similar to
Greiner Bio-one UV-Star 384 well plate (Cat no 781801) (Normal 384) before & after reagent addition.
Greiner Bio-one Clear 384 well plate (Cat no 788876) (Low volume 384) the unknown protein
The plates were incubated for 5 min at RT and measured at 660 nm (Multiskan
GO) Especially in the direct method the sample volume (pathlength) is
Instruments The reported assay linear range is 50-2,000g/mL on a microplate. critical
Thermo Scientific Multiskan GO microplate spectrophotometer (Cat no
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 252000 g/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 cant exactly be expected.
.