BSA Thermo

y = 783.62x - 26.739
R = 0.9963
0
300
600
0 0.4 0.8
u
g
/
m
l
A595
BSA Thermo
y = 289.93x2 + 548.65x + 4.0429
R = 1
0
250
500
0 0.4 0.8
u
g
/
m
l
A562
Coomassie-stained SDS-PAGE gel
to verify protein extraction and
digestion
Determining the Most Accurate Assay
for Measuring Peptide Concentration
Jeneal Carter, Janice L. Hallows, Robert L. Moritz
Individual Assays
and Results
Conclusions
Abstract
NanoDrop (A
280
)
dotMETRIC BCA (A
562
)
OPA (360/460) Bradford (A
595
)
Pipette 1ul of sample onto reading plate, close arm, and take
measurements.

Make sample dilutions in HEPES to reach 0.5 mg/ml, then mix with
dotMETRIC buffer. Apply 1ul spots to strips in triplicate, using
capillaries. Fix and develop, then measure.

Add 20ul sample + 200ul OPA reagent in an opaque 96-well plate.
Measure fluorescence at excitation 360nm and emission 460nm
within 1-5 minutes of mixing.
Mix 5ml dye + 25ml MQ H
2
O and filter to make reagent. Add 160ul
sample + 40ul reagent in a 96-well plate. Incubate at RT x 5min.
Measure absorbance at 595nm.

Mix 9000ul A + 180ul B to make working reagent. Add 25ul sample
+ 200ul WR in a 96-well plate. Mix x 30sec. Cover, incubate at 37
o
C
x 30min. Cool to RT, measure absorbance at 562nm.
A variety of assays are commonly used to determine
protein concentration, however, an effective assay to
determine peptide concentration has not yet been
established. Peptide samples present challenges that
protein samples do not:

Peptides typically do not contain the specific
amino acid residues (e.g. tyrosine and tryptophan)
that are recognized by the assay.
-
The standards used for determining protein
concentration (e.g. a protein solution of known
concentration) do not perform well with peptides.
-
In order to determine which assay most accurately
measures peptide concentration, we prepared a series
of standards (25-500 ug/ml) using 5 different types of
starting material:
1. Commercially available BSA solution (Thermo
Scientific)
2. BSA solution prepared in the lab (concentration
determined by A280/extinction coefficient (e))
3. Whole cell lysate of the HCT-15 colon cancer cell
line (concentration determined by BCA assay)
4. BSA peptides produced by trypsin digestion of the
BSA solution (#2 above)
5. HCT-15 peptides produced by trypsin digestion of
the HCT-15 lysate (#3 above)
The five 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.

lyse cells
extract protein
reduce
alkylate
digest
peptide standards
Assay Method Interfering Agents Calculations Misc. Dynamic Range
BCA (A562)
Thermo
Scientific
Colorimetric:
Bicinchoninic acid (BCA) detects for Cu+1
(formed when Cu2+ is reduced by protein in
an alkaline environment) yielding a purple-
colored reaction product.
Ascorbic Acid
Catecholamines
Creatinine
Cysteine
EGTA
Impure Glycerol
Hydrogen
Peroxide
Hydrazides
Iron
Lipids
Melibiose
Phenol Red
Impure Sucrose
Tryptophan
Tyrosine
Uric Acids
standard curve detergent
compatible

fairly stable
under alkaline
conditions
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
SodiumHydroxide
Basic Protein Buffers
standard curve 2x as much
protein-to-
protein variation
than BCA
50-500 ug/ml
NanoDrop
(A280)
Thermo
Scientific
Protein in solution absorbs UV light at a
wavelength of 280nm, due to the presence
of aromatic amino acids, mainly tyrosine
and tryptophan.
Any non-protein component that absorbs
ultraviolet light e.g.:
Beers Law
(multiply by
extinction
coefficient)
considerable
error for protein
mixtures
100-100,000
ug/ml
Nucleotides
Nucleic Acids
dotMETRIC
GBiosciences
Diameter of protein spots is proportional
to their protein concentration.
Resistant to most common laboratory agents. measure dots
using
exponential
scale
no protein-to-
protein variation
0.031-2.0 ug/ml
OPA
(360/460)
Thermo
Scientific
o-phthalaldehyde (OPA) reacts with the
primary amines of the protein in the
presence of mercaptoethanol, yielding a
blue-colored fluorescent product which
has a maximumwavelength of excitation of
340nmand emission at 455nm.
Amine-containing buffers linear trend
line
compatible with
detergents and
reducing agents
.050 ug/ml-
25ug/ml
Figure 2: developed spots
A1 0.051 A1 0.039 A1 0.100
A2 0.054 A2 0.039 A2 0.063
A3 0.047 A3 n/a A3 0.044
avg 0.051 avg 0.039 avg 0.069
conc (*6 dil) 0.304 conc (*6 dil) 0.234 conc (*6 dil) 0.414
A1 0.051 A1
A2 0.047 A2
A3 0.054 A3
avg 0.051
conc (*6 dil) 0.304
A1 0.100 A1
A2 0.083 A2
A3 0.077 A3
avg 0.087
conc (*3 dil) 0.260
not visible
not visible
JC BSA (9.741) BSA peptides (2) BSA Pierce (2)
HCT-15 (10.11) HCT-15 peptides (2)
liver digest (0.605) amino acids (.137)
A1 5.913 A1 1.187 A1 1.390
A2 5.960 A2 1.135 A2 1.380
A3 5.936 A3 1.149 A3 1.399
avg 5.936 avg 1.157 avg 1.390
0.667 0.667 0.667
conc 8.900 conc 1.735 conc 2.083
A1 29.617 A1 5.752
A2 29.748 A2 5.253
A3 29.402 A3 5.133
avg 29.589 avg 5.379
A1 0.907 A1 0.348
A2 0.927 A2 0.315
A3 0.905 A3 0.330
avg 0.913 avg 0.331
liver digest (0.5) amino acids (.137)
JC BSA (9.741) BSA peptides (2) BSA Pierce (2)
HCT-15 (10.11) HCT-15 peptides (2)
Methods
"Unknowns" BSA Thermo JC BSA BSA peptides HCT-15 lysate HCT-15 peptides
BSA Thermo (0.5mg/ml) n/a 0.471 0.518 0.444 0.423
JC BSA (0.5mg/ml) 0.535 n/a 0.549 0.470 0.449
BSA peptides (0.5mg/ml) 0.483 0.450 n/a 0.425 0.404
HCT-15 lysate (0.5mg/ml) 0.567 0.529 0.581 n/a 0.477
HCT-15 peptides (0.5mg/ml) 0.589 0.550 0.604 0.517 n/a
Standards
"Unknowns" BSA Thermo JC BSA BSA peptides HCT-15 lysate HCT-15 peptides
BSA Thermo (0.5mg/ml) n/a 0.501 2.635 0.681 3.263
JC BSA (0.5mg/ml) 0.474 n/a 2.547 0.658 3.152
BSA peptides (0.5mg/ml) 0.067 0.042 n/a 0.114 0.587
HCT-15 lysate (0.5mg/ml) 0.118 0.098 0.745 n/a 0.913
HCT-15 peptides (0.5mg/ml) 0.354 0.352 1.937 0.497 n/a
Standards
BSA Thermo
y = 0.0241x + 4.1095
R = 0.9993
0
250
500
0 10000 20000
u
g
/
m
l
360/460
Table 1: Individual protein assays

Figure 1: Preparation of peptide standards

BSA Thermo JC BSA BSA peptides HCT-15 lysate HCT-15 peptides
BSA Thermo (0.5mg/ml) n/a 0.430 0.598 0.497 0.701
JC BSA (0.5mg/ml) 0.577 n/a 0.696 0.571 0.843
BSA peptides (0.5mg/ml) 0.425 0.361 n/a 0.422 0.566
HCT-15 lysate (0.5mg/ml) 0.503 0.433 0.602 n/a 0.707
HCT-15 peptides (0.5mg/ml) 0.386 0.325 0.452 0.383 n/a
Standards
"Unknowns"
Assay Advantages Disadvantages Works with
Peptides?
BCA (A562) Easy
Good sensitivity for proteins
Dependent on type of
standard
No
Bradford (A595) Very quick Not as sensitive No
NanoDrop (A280) Works well for individual
protein but not complex
mixtures
Large variability even for
single protein solutions
No
dotMETRIC No interfering agents Low reproducibility
Technically difficult
Measuring spots is subjective
No
OPA Quick
Reproducible
Not dependent on type of
standard used
Very sensitive to air bubbles Yes
Table 2: Results

Funding