Application Note – Industrial BioDevelopment Laboratory (www.ibdl.

ca)
Comparison of 4 Protein Analysis Methods: Kjeldahl, Bradford, Lowry, and Bicinchoninic Acid (BCA) assays
Andrew Wong, Andre C. Siegel, Justin Manuel, Gary A. Levy

Purpose: The purpose of this study was to compare 4 different

standard protein analysis techniques – Bradford, Lowry, BCA, and
Kjeldahl – with regards to their sensitivity, accuracy, and ease of
performance.

Introduction: The various protein analysis techniques available are

all uniquely dependent on protein composition – amino acid content
and any covalently bound material – and protein quantity. Hence, it
is important that the standard assay of choice be appropriate to the
particular sample. This report covers only the Bradford, Lowry,
BCA, and the Kjeldahl method out of a plethora of other protein
assays. The Bradford assay involves the binding of the Coomassie
Brilliant Blue G-250 dye to protein. The dye binds more favourably
to basic residues like arginine. The Lowry assay uses the reaction of Figure 2. Standard curve for BCA assay
cupric ions and protein which forms a complex. This complex later
reduces the Folin-Ciocalteu reagent resulting in a blue colour. The
BCA assay involves the reduction of cupric ions to cuprous ions by
protein – cysteine, cystine, tyrosine, and tryptophan reduces Cu +2
more favourably – such that BCA chelates with the cuprous ions to
form a purple complex. Therefore, different methods will yield slight
variations when quantifying proteins. On the contrary, the Kjeldahl
method measures only total organic nitrogen content and is not
affected by amino acid content. Thus, the Kjeldahl method measures
absolute concentration and is considered the ‘gold standard’ in
protein quantification.

Methodology: BSA (bovine serum albumin), human serum

immunoglobulin G (IgG), and human Fc–FGL2 (Fc tagged
fibrinogen-like protein 2) were each analyzed with the 4 different Figure 3. Standard curve for Lowry assay
protein assays. In all cases, standard curves were constructed using
BSA standards (1 – 1500 µg/mL). The starting concentration of Table 1. Comparison of Different Protein Assays Ability to Determine Protein
BSA, IgG, and Fc-FGL2 used was approximately 500 µg/mL. The Concentration for BSA, IgG, Fc-FGL2
Bradford assay was only linear within 1-125 µg/mL range. BSA IgG Fc-FGL2
Therefore, protein samples required a 10-fold dilution to (µg/mL) (µg/mL) (µg/mL)
approximately 50 µg/mL Average of Duplicate Analyses
Bradford 848 545 752
Results: S tandard C urve for B radford A s s ay
Bradford (with 434 124 140
1.2 10-fold dilution)
A bs orbanc e @ 620nm

1 Lowry 546 478 481

0.8
0.6 BCA 536 480 469
y = 0.00060x + 0.14157
0.4 2
R = 0.81556
0.2 Kjedahl 519 305 322
0
-0.2 0 200 400 600 800 1000 1200 1400 1600

C onc entration (µg /mL ) Conclusion: All 3 colourimetric assays gave slight variations in
S tandard C urve for B radford A s s ay protein concentrations, regardless of the constant starting
concentration. This was expected, due to differences in amino acid
0.35 content of BSA, IgG, and Fc-FGL2 and the mechanisms behind the
A bs orbanc e @ 620nm

0.3 assays. In comparison to the absolute values determined by Kjeldahl

0.25
method, BCA remained the most reliable protein assay when
0.2
quantifying BSA, IgG, and Fc-FGL2. The Lowry assay showed
0.15
0.1
y = 0.0025x - 0.0026
2
comparable sensitivity with the BCA assay. The Bradford assay was
R = 0.9809
0.05
least reliable.
0 The use of human serum IgG in Kjeldahl had ambiguity
-0.05 0 20 40 60 80 100 120 140 due to the multiple isotypes that exist for IgG. Constant nitrogen
C onc entration (µg /mL )
content could not be determined and therefore, the protein
concentration determined was only approximate.
Figure 1. Standard curve for Bradford assay in the 1 – 1500 µg/mL and 1 –
125 µg/mL range respectively.