Determining

the Most Accurate Assay

for Measuring Pep6de Concentra6on
Jeneal Carter, Janice L. Hallows, Robert L. Moritz

Individual Assays
and Results

Figure 1: Prepara<on of pep<de standards

lyse cells

BSA Thermo (0.5mg/ml)

JC BSA (0.5mg/ml)
BSA peptides (0.5mg/ml)
HCT-15 lysate (0.5mg/ml)
HCT-15 peptides (0.5mg/ml)

extract protein

2.635
2.547
n/a
0.745
1.937

ug/ml

600

Thermo
ScienVc

3.263
3.152
0.587
0.913
n/a

Coomassie-stained SDS-PAGE gel

to verify protein extrac<on and
diges<on
Calcula6ons

Misc.

Dynamic Range
20-2000 ug/ml

Bradford
(A595)
BioRad

Colorimetric:
Dye turns from reddish/brown to blue as
protein binds to the coomassie dye in the
acidic environment of the reagent.

Some Detergents
Flavonoids
Sodium Hydroxide
Basic Protein Buers

standard curve 2x as much

50-500 ug/ml
protein-to-
protein variaVon
than BCA

NanoDrop
(A280)
Thermo
ScienVc
dotMETRIC
GBiosciences

Protein in soluVon absorbs UV light at a

wavelength of 280nm, due to the presence
of aromaVc amino acids, mainly tyrosine
and tryptophan.

Any non-protein component that absorbs

ultraviolet light e.g.:
NucleoVdes
Nucleic Acids

Beers Law
(mulVply by
exVncVon
coecient)

considerable
100-100,000 ug/
error for protein ml
mixtures

Diameter of protein spots is proporVonal

to their protein concentraVon.

Resistant to most common laboratory agents.

no protein-to-
0.031-2.0 ug/ml
protein variaVon

OPA
(360/460)
Thermo
ScienVc

o-phthalaldehyde (OPA) reacts with the

primary amines of the protein in the
presence of mercaptoethanol, yielding a
blue-colored uorescent product which
has a maximum wavelength of excitaVon
of 340nm and emission at 455nm.

Amine-containing buers

measure dots
using
exponenVal
scale
linear trend
line

500

y = 783.62x - 26.739
R = 0.9963

300

0.4

JC BSA (9.741)
A1
5.913
A2
5.960
A3
5.936
avg
5.936

0.667
conc
8.900

BSA peptides (2)

A1
1.187
A2
1.135
A3
1.149
avg
1.157

0.667
conc
1.735

HCT-15 (10.11)
A1
29.617
A2
29.748
A3
29.402
avg
29.589

HCT-15 peptides (2)

A1
5.752
A2
5.253
A3
5.133
avg
5.379

liver digest (0.5)

A1
0.907
A2
0.927
A3
0.905
avg
0.913

amino acids (.137)

A1
0.348
A2
0.315
A3
0.330
avg
0.331

0.4
A562

0.8

0.051
0.054
0.047
0.051
0.304

A1
A2
A3
avg
conc (*6 dil)

0.039
0.039
n/a
0.039
0.234

BSA Pierce (2)

A1
1.390
A2
1.380
A3
1.399
avg
1.390

0.667
conc
2.083

10000

20000

HCT-15 peptides (2)

A1
A2
A3

liver digest (0.605)

A1
0.100
A2
0.083
A3
0.077
avg
0.087
conc (*3 dil)
0.260

amino acids (.137)

A1
A2
A3

A1
A2
A3
avg
conc (*6 dil)

Table 2: Results

Assay

Advantages

Easy
Good sensiVvity for proteins
Bradford (A595) Very quick
NanoDrop (A280) Works well for individual
protein but not complex
mixtures
dotMETRIC
No interfering agents

0.100
0.063
0.044
0.069
0.414

ib
l

vi
s

Conclusions
BCA (A562)

HCT-15 (10.11)
A1
0.051
A2
0.047
A3
0.054
avg
0.051
conc (*6 dil)
0.304

ib
l

0.701
0.843
0.566
0.707
n/a

A1
A2
A3
avg
conc (*6 dil)

vi
s

y = 289.93x2 + 548.65x + 4.0429

R = 1

250

360/460

PipeQe 1ul of sample onto reading plate, close arm, and take
measurements.

compaVble with .050 ug/ml-25ug/

detergents and ml
reducing agents

0.497
0.571
0.422
n/a
0.383

BSA Thermo

250

y = 0.0241x + 4.1095
R = 0.9993

0.8

dotMETRIC

0.598
0.696
n/a
0.602
0.452

HCT-15 peptides
0.423
0.449
0.404
0.477
n/a

no
t

0.430
n/a
0.361
0.433
0.325

HCT-15 lysate
0.444
0.470
0.425
n/a
0.517

BSA Thermo

Mix 9000ul A + 180ul B to make working reagent. Add 25ul sample Make sample diluVons in HEPES to reach 0.5 mg/ml, then mix with
+ 200ul WR in a 96-well plate. Mix x 30sec. Cover, incubate at 37oC dotMETRIC buer. Apply 1ul spots to strips in triplicate, using
x 30min. Cool to RT, measure absorbance at 562nm.
capillaries. Fix and develop, then measure.
Standards
JC BSA (9.741)
BSA peptides (2)
BSA Pierce (2)

"Unknowns"
BSA Thermo
JC BSA
BSA peptides
HCT-15 lysate
HCT-15 peptides

500

JC BSA
0.471
n/a
0.450
0.529
0.550

Standards
BSA peptides
0.518
0.549
n/a
0.581
0.604

NanoDrop (A280)

BCA (A562)

n/a
0.577
0.425
0.503
0.386

BSA Thermo
n/a
0.535
0.483
0.567
0.589

A595

standard curve detergent

Bicinchoninic acid (BCA) detects for Cu+1
Catecholamines Peroxide

Impure Sucrose
compaVble
(formed when Cu2+ is reduced by protein in CreaVnine
Hydrazides Tryptophan

an alkaline environment) yielding a purple- Cysteine
Iron
Tyrosine
fairly stable
colored reacVon product.
EGTA
Lipids
Uric Acids
under alkaline
Impure Glycerol Melibiose
condiVons

BSA Thermo (0.5mg/ml)

JC BSA (0.5mg/ml)
BSA peptides (0.5mg/ml)
HCT-15 lysate (0.5mg/ml)
HCT-15 peptides (0.5mg/ml)

"Unknowns"
BSA Thermo (0.5mg/ml)
JC BSA (0.5mg/ml)
BSA peptides (0.5mg/ml)
HCT-15 lysate (0.5mg/ml)
HCT-15 peptides (0.5mg/ml)

Table 1: Individual protein assays

Method
Interfering Agents
BCA Assay
Colorimetric:
Ascorbic Acid Hydrogen

Phenol Red
(A )
562

0.681
0.658
0.114
n/a
0.497

Add 20ul sample + 200ul OPA reagent in an opaque 96-well plate.

Measure uorescence at excitaVon 360nm and emission 460nm
within 1-5 minutes of mixing.

BSA Thermo

pep6de standards

ug/ml

In order to determine which assay most accurately

measures pepVde concentraVon, we prepared a series
of standards (25-500 ug/ml) using 5 dierent types of
starVng material:
1. Commercially available BSA soluVon (Thermo
ScienVc)
2. BSA soluVon prepared in the lab (concentraVon
determined by A280/exVncVon coecient ())
3. Whole cell lysate of the HCT-15 colon cancer cell
line (concentraVon determined by BCA assay)
4. BSA pepVdes produced by trypsin digesVon of the
BSA soluVon (#2 above)
5. HCT-15 pepVdes produced by trypsin digesVon of
the HCT-15 lysate (#3 above)
The ve protein assays listed in Table 1 were
performed using all 5 sets of standards, and the 500
ug/ml standard from each set was arbitrarily chosen to
serve as the unknown for the assays.

0.501
n/a
0.042
0.098
0.352

Methods

n/a
0.474
0.067
0.118
0.354

reduce
alkylate
digest

OPA (360/460)

Mix 5ml dye + 25ml MQ H2O and lter to make reagent. Add 160ul
sample + 40ul reagent in a 96-well plate. Incubate at RT x 5min.
Measure absorbance at 595nm.
Standards

"Unknowns"
BSA Thermo
JC BSA
BSA peptides
HCT-15 lysate
HCT-15 peptides

no
t

A variety of assays are commonly used to determine

protein concentraVon, however, an eecVve assay to
determine pepVde concentraVon has not yet been
established. PepVde samples present challenges that
protein samples do not:
PepVdes typically do not contain the specic
amino acid residues (e.g. tyrosine and tryptophan)
that are recognized by the assay.
The standards used for determining protein
concentraVon (e.g. a protein soluVon of known
concentraVon) do not perform well with pepVdes.

Bradford (A595)

ug/ml

Abstract

Figure 2: developed spots

OPA

Quick
Reproducible
Not dependent on type of
standard used

Disadvantages
Dependent on type of
standard
Not as sensiVve
Large variability even for
single protein soluVons
Low reproducibility
Technically dicult
Measuring spots is subjecVve
Very sensiVve to air bubbles

Funding

Works with
Pep6des?
No
No
No

No

Yes