Bioseparation Process II

Dr. Munira Shahbuddin

Chapter 2 and 3 Cell lysis

Bicinchoninic Acid (BCA) Method Protein Assay

Introduction
Bichichonic Acid (BCA) Protein assay is a colorimetric method that involves protein-copper chelation
with secondary detection of the reduced copper binding to protein. The binding of protein to reduced
copper ions will give a coloration change at 540 nm wavelength. This copper based assay chemistry is
also well known as biuret reaction. In this reaction, biuret, which is a product of excess urea and
heat reacts with copper and reduce the 2+ ions to cuprous ions which form a light blue tertadentate
complex.

Figure 1 .Structures of urea, biuret and peptide. The similarity of polypeptide structures to biuret and
urea makes it able to reduce copper ions via biuret reaction.

BCA has become the most popular method for calorimetric detection and quantification of protein
ever since it was introduced by Smith et. al in 1985. BCA assay is similar to Lowry method which
both are well known to be dependent on the reduction of Cu2+ to Cu1+ under alkaline condition as
described in Biuret reaction. Then this cuprious cation Cu1+ will be chelated by bichinconinic acid
(BCA), resulting in an intense purple color. The purple color product is formed by the chelation of
two BCA molecules with one cuprous ion as in figure 1. The purple color will become intense with
increasing protein concentration which can be measured at any wavelength between 550nm to 570nm.
In this reaction, single amino acids and dipeptides do not give the changes in coloration to violet
complex as the reaction is more sensitive to tripeptides, larger polypeptides and proteins. The

BCA/copper complex exhibits strong linear absorbance at 562nm with increasing protein
concentration.

The rate of BCA color formation is also dependent on the incubation temperature and time. In order to
obtain accurate results with BCA assay method is to assay standard and unknown samples
simultaneously so they both receive identical incubation time and temperature.

Figure 2.Diagram of the biuret reaction. Reduction of copper ion from cupric to cuprous produces a
faint blue violet color.

Figure 3.The reaction of BCA with cupric ion. Two molecules of BCA bind to coprious ion that had
been reduced by peptide mediated biuret reaction

Materials
Pierce BCA Protein Assay was obtained from Thermo Scientific (Rockford, IL USA) and used
without further purification.

Methods
Standard protein solutions were prepared by diluting a 2mg/ml BSA stock standard in the extraction
solution as in table 2. The stock solution is provided in ampules of 1ml. The working reagent was
prepared by mixing reagent A with reagent B in a ratio of 50:1, to create a clear green solution. 25l
of each extraction solution and standard solution was added to individual wells in a new 96-well plate.
Three blanks were also prepared using neat extraction solution. To each well 200l of the working
reagent was added and the plate was then placed on a shaker 30 seconds and incubated at 37C for
30minutes. The plate was allowed to cool to room temperature, and the absorbance was then read at
540nm using a Dynex MRX Revelation 96 Well Microplate Reader.
Table 2 Preparation of BSA standard solutions
Standard number

1
2
3
4
5
6
7
8

BSA added /l

300 of stock
375 of stock
325 of stock
175 of 2
325 of 3
325 of 5
325 of 6
100 of 7

Diluent added / l

0
125
325
175
325
325
325
400

Final BSA
concentration
/gml-1
2000
1500
1000
750
500
250
125
25

Figure 4. Schematic diagram of the experiment on protein absorption in KGM hydrogels and
detection using BCA calorimetric assay.

MTT assay.

Measurement of cell viability can be assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) MTT assay. MTT is a colorimetric method used to measure the
metabolic activity of viable cells. When in contact with MTT, metabolically active cells will reduce
the chemical into formazan dye, giving a purple color. This formazan can be extracted and dissolved
in isopropanol and the absorbance can be quantified using a spectrometry at absorbance maximum
460-540 nm.

Figure 5

Reduction of MTT by mitochondrial dehydrogenase.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

or

MTT

assay

is

colourimetric indicator of cell number or viability by means of mitochondrial dehydrogenase

activity according to method of Nik and Otto (Nik and Otto 1990). The cells were gently
washed with PBS and 1.0 mL of MTT solution was added per well. Then the plates were
incubated with for 40 min at 37 oC, 5 % CO2 in a humidified atmosphere. During this time,
mitochondrial dehydrogenase activity will reduce MTT into an insoluble purple-coloured
formazan product that can be eluted using acidified isopropanol, made of 0.125 L1 M HCl
in 100 mL of isopropanol. After 40 min, the MTT solution was subsequently removed and
200 l of acidified isopropanol was used to elute the formazan product from the cells. 200 l
of the isopropanol was then transferred into a 96 well plate and the optical density at 540 nm
(and reference at 630 nm) was read in a Dynex Technologies MRXII microplate reader
attached to a PC running Revelation 2.0 software.

AlamarBlue assay

Figure 6

Reduction of Resazurin by mitochondrial dehydrogenase

AlamarBlue assay is a colourimetric growth indicator based on detection of metabolic

activity. The redox indicator in the solution changes colour from blue to red in response to
chemical reduction of alamar blue into purple by the mitochondrial dehydrogenase activity in
cell nuclei. The changes in the colour which indicate the number of cells or cell viability was
then measured using a plate reader. To measure the cell viability, first the samples were
washed with PBS and an AlamarBlue assay performed by adding 5 mL of AlamarBlue
(diluted 1:10 in PBS) (AbD Serotec, Kiddlington, UK) and incubated for 60 min. All cultures
were kept in identical conditions of 5% CO2, 37oC, and assayed at the same time point.
Absorbance at 570 nm was then measured in a colourimetric plate reader (Bio-TEK,
NorthStar Scientific LTD, Leeds, UK) to obtain baseline values of cell attachment. Samples
were then washed with PBS and returned to culture conditions. AlamarBlue assay was
repeated after 7 and 14 days of culture.

Picogreen assay
Picogreen a fluorescent nucleic acid stain was used for the quantification of total cellular
dsDNA. The method used in this study was conducted according to Ahn et. al., 1996 (Ahn,
Costa et al. 1996).
All media was removed and cells were washed twice with PBS. Then 200 L of 10%
digesting buffer was added and cells were frozen at -80oC and then thawed in a dry incubator
for three cycles to break the cell membranes and extract the DNA. The cells were then
scraped off and centrifuged at (~1700 g) for 10 min to collect the DNA from the supernatant.
100 L of the supernatant was added to100 L of Picogreen (1:200) and mixed well. 100 L
of this solution was then transferred to a fluorescence plate reader (Biotex instruments, Inc.,
USA) and samples excited at 340 nm with an emission wavelength at 488 nm. A quantitative
estimation of the cell number was obtained by calibrating this reading against a known
number of cells using this method.