Bicinchoninic Acid (BCA)-based Protein Assays

In 1985, Paul K. Smith, et al. introduced the BCA Protein Assay. Since then it has
become the most popular method for colorimetric detection and quantitation of total
protein. The BCA Protein Assay has a unique advantage over the Modified Lowry
Protein Assay and any of the Coomassie dye-based assays it is compatible with
samples that contain up to 5% surfactants (detergents). Briefly, the sample is added to
the tube or plate containing the prepared BCA Working Reagent and after a 30-minute
incubation at 37C and cooling to room temperature, the resultant purple color is
measured at 562nm. The protocol is similar for the Micro BCA Protein Assay, except the
ratio of sample volume to working reagent is different and the tubes are incubated for 60
minutes at 60C
Pada tahun 1985 Paul K. Smith memperkenalkan pengujian Protein dengan metode
BCA. Sejak saat itu metode tersebut menjadi method yang paling terkenal untuk deteksi
protein berdasarkan warna dan perhitungan kadar protein. Metode BCA memiliki
keuntungan yang unik dibandingkan dengan metode lowry yang termodifikasi dan salah
satu tes berbasis pewarna (metode Bradford). Metode ini cocok untuk menguji sampel
yang mengandung lebih dari 5% surfaktan (detergent). Singkatnya, sampel
ditambahkan ke tabung atau piring (palte ) yang telah mengandung atau berisi BCA
Working agent dan setelah 30 menit inkubasi pada 37 0C dan pendinginan pada suhu
kamar, hasilnya berupa warna ungu yang kemudian diukur pada panjang gelombang
562 nm.

Chemistry of BCA-based Protein Assays

The BCA Protein Assay combines the well-known reduction of Cu2+ to Cu 1+ by protein
in an alkaline medium with the highly sensitive and selective colorimetric detection of
the cuprous cation (Cu 1+) by bicinchoninic acid (Figure 1). The first step is the chelation
of copper with protein in an alkaline environment to form a blue colored complex. In this
reaction, known as the biuret reaction, peptides containing three or more amino acid
residues form a colored chelate complex with cupric ions in an alkaline environment
containing sodium potassium tartrate. This became known as the biuret reaction
because a similar complex forms with the organic compound biuret (NH 2-CO-NHCONH2 ) and the cupric ion. Biuret, a product of excess urea and heat, reacts with
copper to form a light blue tetradentate complex (Figure 2). Single amino acids and
dipeptides do not give the biuret reaction, but tri- peptides and larger polypeptides or
proteins will react to produce the light blue to violet complex that absorbs light at
540nm. One cupric ion forms a colored coordination complex with four to six nearby
peptides bonds.
Metode BCA merupakan kombinasi reduksi terkenal dari Cu2+ ke Cu 1+ oleh protein
dalam medium alkali dengan sensitifitas yang tinggi dan selektifitas deteksi warna yang
tinggi dari kation tembaga (Cu1+) oleh BCA . langkah awal dimana khelat tembaga
dengan protein dalam alkali membentuk warna biru kompleks (dikenal sebagai rx
biuret). Peptida yang mengandung tiga atau lebih asam amino residu membentuk

kompleks khelat berwarna dengan ion tembaga dalam suasana alkali yang
mengandung natrium kalium tartrat. Di kenal sebgai rx biuret karena bentuk kompleks
nya serupa dengan biuret senyawa organic (NH2-CO-NH-CONH2) dan ion tembaga.
Biuret merupakan sebuah produk urea berlebih dan panas. Bereaksi dengan tembaga
untuk membentuk kompleks cahaya biru tetradentate. Satu asam amino atau dipeptida
tidak bereaksi positif tetapi tripeptida atau polipeptida atau protein akan bereaksi
dengan membentuk warna kompleks biru sampai ungu yang menyerap cahaya pada
panjang gelombang 540 nm. Satu ion tembaga membentuk senyawa kompleks
koordinasi berwarna dengan 4 atau 6 ikatan peptide didekatnya
The intensity of the color produced is proportional to the number of peptide bonds
participating in the reaction. Thus, the biuret reaction is the basis for a simple and rapid
colorimetric reagent of the same name for quantitatively determining total protein con
centration. Since the working range for the biuret assay is from 5 to 160mg/mL, the
biuret assay is used in clinical laboratories for the quantitation of total protein in serum
Intensitas warna yang dihasilkan sebanding dengan jumlah ikatan peptide yang
berpartisipasi dalam reaksi . Jadi, reaksi biuret merupakan dasar untuk reagen
colorimetric yang sederhana untuk penentuan kuantitatif kadar konsentrasi protein.
Karena kisaran untuk Metode Biuret adalah dari 5 sampai 160 mg/mL, maka biasanya
Metode biuret digunakan untuk lab pengobatan untuk mengukur kadar proteindalam
serum secara kuantitatif.

In the second step of the color development reaction, BCA Reagent, a highly sensitive
and selective colorimetric detection reagent reacts with the cuprous cation (Cu 1+) that
was formed in step 1. The purple colored reaction product is formed by the chelation of
two molecules of BCA Reagent with one cuprous ion (Figure 1). The BCA/Copper
Complex is water-soluble and exhibits a strong linear absorbance at 562nm with
increasing protein concentrations. The purple color may be measured at any
wavelength between 550-570nm with minimal (less than 10%) loss of signal. The BCA
Reagent is approximately 100 times more sensitive (lower limit of detection) than the
biuret reagent.
Pada langkah kedua dari reaksi berwarna, reagen BCA merupakan reagen yang
sensitifitas dan selektifitas warnanya (colorimetric) sangat tinggi bereaksi dengan
kation tembaga (Cu 1+) yang terbentuk pada langkah awal. Produk warna ungu
terbentuk dari dua molekul khelat Reagen BCA dengan satu ion tembaga. BCA/
kompleks tembaga larut dalam air dan menunjukkan absorbansi linier yag kuat pada
562nm dengan meningkatnya konsentrasi protein. Warna ungu memungkinkan dapat
diukur pada setiap gelombang antara 550-570nm. Reagen BCA 100 kali lebih sensitive
dibanidng reagen biuret

The reaction that leads to BCA Color Formation as a result of the reduction of Cu 2+ is
also strongly influenced by the presence of any of four amino acid residues (cysteine or
cystine, tyrosine, and tryptophan) in the amino acid sequence of the protein. Unlike the
Coomassie dye-binding methods that require a minimum mass of protein to be present
for the dye to bind, the presence of only a single amino acid residue in the sample may
result in the formation of a colored BCA-Cu1+ Chelate. This is true for any of the four
amino acids cited above. Studies performed with di- and tripeptides indicate that the
total amount of color produced is greater than can be accounted for by the color
produced with each BCA Reagent-reactive amino acid. Therefore, the peptide
backbone must contribute to the reduction of copper as well.The rate of BCA Color
Formation is dependent on the incuba-tion temperature, the types of protein present in
the sample and the relative amounts of reactive amino acids contained in the proteins.
The recommended protocols do not result in end-point determinations, the incubation
periods were chosen to yield maximal color response in a reasonable time frame.

Advantages of the BCA Protein Assay

The primary advantage of the BCA Protein Assay is that most surfactants (even if
present in the sample at concentrations up to 5%) are compatible. The protein:protein
variation in the amount of color produced with the BCA Protein Assay is relatively low,
similar to that observed for the Modified Lowry Protein Assay (Table 2, page 9). The
BCA Protein Assay produces a linear response curve (r2 > 0.95) and is available in two
formulations based upon the dynamic range needed to detect the protein concentration
of an unknown sample. The BCA Assay is less complicated to perform than the Lowry
Protein Assay for both formulations. The standard BCA Protein Assay (Figure 3) detects
protein concentrations from 20 to 2,000g/mL and is provided with Reagent A
(carbonate buffer containing BCA Reagent) and Reagent B (cupric sulfate solution). A
working solution (WS) is prepared by mixing 50 parts of BCA Reagent A with 1 part of
BCA Reagent B (50:1, Reagent A:B). The working solution is an apple green color that
turns purple after 30 minutes at 37C in the presence of protein. The ratio of sample to
WS used is 1:20. The Micro BCA Protein Assay (Figure 4) is more sensitive and has a
narrower dynamic range of 0.1-25g/mL. To prepare the Micro BCA WS, three reagents
(A, B and C) are mixed together at a ratio of 25 parts Micro Reagent A to 24 parts Micro
Reagent B and 1 part Micro Reagent C. The Micro BCA WS is mixed with the sample or
standard at a 1:1 volume ratio. The purple color response is read at 562nm after 1 hour
at 60C. Since the color reaction is not a true end-point reaction, consid-erable protocol
flexibility is allowed with the BCA Protein Assay. By increasing the incubation
temperature, the sensitivity of the assay can be increased. When using the enhanced
tube pro-tocol (incubating at 60C for 30 minutes), the working range for the assay shifts
to 5-250g/mL and the minimum detection level becomes 5g/mL.
Both BCA Protein Assay formulations have less protein:protein variability than the
Coomassie-based assays. The color response obtained for a seven point standard
curve with the standard BCA Protein Assay using BSA or BGG standards shows less

than a 20% variation between these two proteins (Figure 3). The Coomassie assay
demonstrates >30% variation in the signal generated between BSA and BGG (Table 2,
page 9). There is even less variation (<12%) when comparing these protein standards
with the Micro BCA Protein Assay (Figure 4). In general, the BCA Protein Assay
provides one of the most accurate measurements of protein concentration in biological
samples, is detergent-compatible and simple to perform

Disadvantages of the BCA Protein Assay

Substances that reduce copper will also produce color in the BCA Assay, thus
interfering with the accuracy of the protein quantitation. Reagents that chelate the
copper also interfere by reducing the amount of BCA color produced with protein.
Certain single amino acids (cysteine or cystine, tyrosine and tryptophan) will also
produce color and interfere in BCA Assays.

BCA Protein Assay Reducing Agent Compatible

The Thermo Scientific Pierce BCA Assay is always compatible with more detergents,
buffers/salts and solvents than any other colorimetric protein assay. Now its
compatiblewith reducing agents at concentrations routinely used in protein sample
buffers!
The BCA Assay provides one of the most accurate measure-ments of protein
concentration in biological samples available. Although the BCA Assay is compatible
with more detergents, buffers/salts and solvents than any colorimetric protein assay, the
presence of disulfide reducing agents, including dithiothreitol (DTT) and 2
mercaptoethanol interferes with the assay. The BCA Protein Assay Kit Reducing
Agent Compatible (Product # 23250) provides all the advantages of the original BCA
Assay as well as compatibility with reducing agents at concentrations routinely used in
protein sample buffers (Figures 1 and 2).
Highlights:
Compatible with up to 5mM DTT, 35mM 2-mercaptoethanol or
10mM TCEP
No protein precipitation required
Linear working range: 125-2,000g/mL
Sample volume: 25L
Compatible with most ionic and nonionic detergents
Significantly less protein:protein variation than coomassie
(Bradford)-based methods
Colorimetric method; measure at 562nm
Easy-to-use protocol (Figure 2)

The Original BCA Protein Assay

Used in more labs than any other detergent-compatible formulation
Highlights:

Colorimetric method; read at 562nm

Compatible with most ionic and nonionic detergents
Four times faster and easier than the classical Lowry method
All reagents stable at room temperature for two years
Working reagent stable for 24 hours
Linear working range for BSA from 20 to 2,000g/mL
Minimum detection level of 5g/mL with the enhanced protocol
Convenient microplate or cuvette format
Less protein:protein variation than dye-binding methods

Micro BCA Protein Assay

Most sensitive BCA formulation measuring dilute protein
solutions from 0.5 to 20g/mL.
Highlights:
Colorimetric method; read at 562nm
Compatible with most ionic and nonionic detergents
A very sensitive reagent for dilute protein samples
Linear working range for BSA: 0.5-20g/mL
Less protein:protein variation than dye-binding methods
All kit reagents stable at room temperature for two years
Working reagent is stable for 24 hours
Convenient microplate or cuvette format

Bicinchoninic Acid (BCA) Protein Assay (Smith)

Considerations for use
The bicinchoninic acid (BCA) assay is available in kit form from Pierce (Rockford, Ill.). This
procedure is very applicable to microtiter plate methods. The BCA is used for the same reasons
the Lowry is used. Stoscheck (1990) has suggested that the BCA assay will replace the Lowry
because it requires a single step, and the color reagent is stable under alkaline conditions.
Metode BCA tersedia dalam bentuk kit (kotak) dari Pierce. Dalam pelaksanaanya menggunakan
metode piring mikro. BCA sama seperti Lowry. Stoscheck menyarankan bahwa metode BCA
akan menggantikan metode lowry karena metode BCA hanya membutuhkan satu langkah kerja.
Selain itu warna reagennya stabil dalam pengaruh alkali.

The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the
relationship between protein content and absorbance is nearly linear over a wide range; however,
performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in
opaque, white 384-well plates. These plates emit fluorescence between 450600 nm when excited at
430 nm; thus, their fluorescence is quenched by the BCA chromophore (max 562 nm). This arrangement
allowed accurate determination of protein content using only 2 L of sample. Moreover, soluble
flourescein could replace the white plates, creating a homogenous format.
Metode BCA merupakan metode yang didasarkan oleh warna untuk penentuan konsentrasi proteinnya.
Hubungan antara kandungan protein dan absorbansi adalah sangat dekat dalam range yang besar.
Meskipun begitu hasilnya akan berkurang seiring berkurangnya volume.

Principle
BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes
between copper ions and peptide bonds to produce a purple end product. The advantage of BCA
is that the reagent is fairly stable under alkaline conditions, and can be included in the copper
solution to allow a one step procedure. A molybdenum/tungsten blue product is produced as with
the Lowry.
Asam bicinchoninic ( BCA ) assay tersedia dalam bentuk kit dari Pierce ( Rockford , Illinois . ) . Prosedur
ini sangat berlaku untuk metode plat mikrotiter . BCA yang digunakan untuk alasan yang sama Lowry
digunakan . Stoscheck (1990 ) telah menyarankan bahwa uji BCA akan menggantikan Lowry karena
memerlukan satu langkah , dan reagen warna stabil pada kondisi basa .
Uji BCA adalah metode kolorimetri untuk memperkirakan konsentrasi protein . Dalam piring 96 - baik ,
hubungan antara kadar protein dan absorbansi hampir linier melalui berbagai ; Namun , kinerja berkurang
dalam volume yang lebih rendah . Untuk mengatasi keterbatasan ini , kami melakukan tes BCA di buram
, putih piring 384 - baik . Lempeng ini memancarkan fluoresensi antara 450-600 nm saat senang pada 430
nm ; dengan demikian , fluoresensi mereka dipadamkan oleh kromofor BCA ( max 562 nm ) .
Pengaturan ini memungkinkan penentuan akurat kandungan protein hanya menggunakan 2 uL sampel .
Selain itu , flourescein larut bisa menggantikan piring putih , menciptakan format homogen .
BCA melayani tujuan reagen Folin dalam uji Lowry , yaitu bereaksi dengan kompleks antara ion tembaga
dan ikatan peptida untuk menghasilkan produk akhir ungu . Keuntungan dari BCA adalah bahwa reagen
cukup stabil pada kondisi basa , dan dapat dimasukkan dalam larutan tembaga untuk memungkinkan
prosedur satu langkah . Sebuah produk biru molibdenum / tungsten diproduksi sebagai dengan Lowry .

Equipment
In addition to standard liquid handling supplies a visible light spectrophotometer is needed with
transmission set to 562 nm. Glass or polystyrene (cheap) cuvettes may be used.

Procedure 1 (standard assay)

Reagents

1. Reagent A: 1 gm sodium bicinchoninate (BCA), 2 gm sodium carbonate, 0.16 gm sodium

tartrate, 0.4 gm NaOH, and 0.95 gm sodium bicarbonate, brought to 100 ml with distilled
water. Adjust the pH to 11.25 with 10 M NaOH.
2. Reagent B: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml distilled water.
3. Standard working solution (SWR): Mix 100 volumes reagent A with 2 volumes reagent
B.
4. The stock solutions are stable. The working solution is stable for 1 week and should be
green.

Assay
1. Prepare samples containing 0.2 to 50 micrograms protein in microliters.
2. Add 1 ml SWR to each 20 microliters sample and mix. Incubate 30 min. at 60 degrees C.
3. Cool the samples and read at 562 nm. Color will be stable for at least one hour.

Procedure 2 (micro assay)

Reagents
1. Reagent A: 8 gm sodium carbonate monohydrate, 1.6 gm sodium tartrate, brought to 100
ml with distilled water. Adjust the pH to 11.25 with 10 M NaOH.
2. Reagent B: 4 gm BCA in 100 ml distilled water.
3. Reagent C: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml water.
4. Working solution: Mix 1 volume reagent C with 25 volumes reagent B, then add 26
volumes reagent A to the C/B mixture.

Assay
1. Prepare samples containing 0.2 to 50 micrograms protein in 500 microliters.
2. Add 500 microliters working solution to each 500 microliters sample and mix. Incubate
60 min. at 60 degrees C.
3. Cool the samples and read at 562 nm.

Analysis
Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine
amounts from the curve. Determine concentrations of original samples from the amount protein,
volume/sample, and dilution factor, if any. If you are unfamiliar with how to obtain a protein
concentration for a diluted sample from a standard curve, see how to prepare and use a protein
standard curve.

Comments

A longer incubation increases the sensitivity of the assay. The heating can be stopped earlier to
prevent the color from becoming too dark. The assay can be performed at room temperature, but
there is greater variability among proteins and the assay is less sensitive.

Reference
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
Protein merupakan unit penyusun utama tubuh. Protein juga merupakan suatu polimer
yang mempunyai monomer suatu asam amino. Asam amino sendiri merupakan senyawa kimia
yang mengandung dua gugus fungsi yang berbeda. Sehingga reaksi identifikasi suatu protein
tidak jauh dari reaksi kedua gugus fungsi tersebut. Salah satu identifikasi protein adalah dengan
cara denaturasi protein (perubahan struktur protein).
Protein merupakan suatu polipeptida dengan BM yang sangat bervariasi dari 5000
samapi lebih dari satu juta karena molekul protein yang besar, protein sangat mudah mengalami
perubahan fisis dan aktivitas biologisnya. Banyak agensia yang menyebabkan perubahan sifat
alamiah dari protein seperti panas, asam, basa, solven organik, garam, logam berat, radiasi sinar
radioaktif (Sudarmadji, 1996).
Sudarmaji, Slamet , dkk. 2007. Analisis bahan Makanan dan Pangan. Penerbit Liberty.

Konsentrasi protein diukur berdasarkan atas optical dencity pada panjang gelombang
tertentu untuk mengetahui banyaknya protein dalam larutan. Protein dengan garam fosfofungsat
pada suasana alkalis akan memberikan warna biru yang intensitasnya tergantung pada
konsentrasi protein tertera. (Arthur, 1996)
Kurva standart adalah kurva Alibrasi dari sederet larutan standart larutan-larutan itu.
Larutan itu sebaiknya mempunyai komposisi cuplikan. Hasil tidak pernah didasarkan pada
literature absortivitas molar. (Polling, 1996)
Analisa Kjeldahl dapat dipakai untuk menganalisis kadar protein dalam bahan makanan
secara tidak langsung. Analisa ini dipakai untuk mengetahui kadar protein dengan menggunakan
asam sulfat pekat dengan katalis selenium oksiklorida. Cara ini merupakan cara yang sederhana
dan mudah dilakukan. (Basari, 1997)
Protein pada setiap bahan kadarnya berbeda-beda. Pengukuran kadar protein suatu bahan
sangat diperlukan karena erat kaitannya dengan tingkat konsumsi manusia. Pengukuran kadar
protein dengan menggunakan metode Lowry adalah dasar dari penggunaan spektrofotometer.
Warna biru yang terjadi oleh pereaksi Ciocalteau disebabkan reaksi antara protein dan Cu dalam
larutan alkalis dan terjadi reaksi garam fosfotungstat dan garam fosfomoliddat oleh tirosin dan
triptopan (Ahmad, 1997)
Protein merupakan makromolekul polipeptida yang tersusun dari sejumlah asam-asam
amino yang dihubungkan oleh ikatan peptida dan mempunyai bobot molekul 5000 sampai
berjuta-juta. Satu molekul protein disusun oleh sejumlah asam amino tertentu dengan susunan
tertentu pula dan bersifat turunan (Aisyah , 1998)
Arthur. 1996. Illustrated Dictionary of Chemistry. Science Press Singapore. Singapore.
Polling. 1996. Intisari Kimia III. UT. Depdikbud. Jakarta.

Basari. 1997. Ilmu Kimia SMU. Depdikbud RI. Jakarta.

Achmad. 1997. Buku Materi Pokok Kimia. UT. Depdikbud RI. Jakarta.
Aisyah. 1998. Kimia Untuk Universitas. Bumi Aksara. Jakarta