Product Description.

High quality reagents for the determination of protein concentration by the bicinchoninic acid method

 

Reference              Name                                                                                                                                                                     

UP75860A       MicroBC Assay protein Quantitation Kit, complete kit qsp 500 (tube) or 3400 (microplate) determinations

                         Contains:          UP67251A            Reagent A, 250ml

                                                  UP67252A            Reagent B, 250ml

                                                  UP67253A            Reagent C, 12ml

                                                  UP36859A            BSA standard, 10x1ml

UP75860C       MicroBC Assay protein Quantitation Kit, complete kit qsp 50 (tube) or 340 (microplate) determinations

                         Contains:          UP67251C            Reagent A, 25ml

                                                  UP67252C            Reagent B, 25ml

                                                  UP67253C            Reagent C, 1.2ml

                                                  UP36859A            BSA standard,1ml

 

Storage: 1 year from receipt, at room temperature (long term storage at + 4°C)

 

For laboratory use only, not for drug, household or medical use

 

Principle:

The MicroBC Assay is a colorimetric assay: it involves the reduction of Cu2+ to Cu+ by peptidic bounds of proteins.     The BC (BicinChoninic acid) chelates Cu+ ions with very high specificity to form a water soluble purple coloured complex. The reaction is increased by high temperatures. As it continues over time, the reaction should be read at a defined time and temperature.

Peptidic bounds  + Cu2+ ------ > [ tetradente- Cu+  complex ]

                                                OH-                         Cu+    +  2 BC --- > [ purple-colored Cu+-BC Assay-Complex ]

 

This reaction is measured by the high optical absorbance of the final Cu+  complex at 562 nm. Absorbance is directly proportionnal to the protein concentration, with a broad linear range between 1 µg/ml to 100 mg/ml. The protein concentration can be calculated with a reference curve obtained for a standard protein.

Assay Procedure

 

Labware must be carefully cleaned and rinced with distilled water to avoid traces of proteins and metals.

 

Preparation of samples:

The protein concentration must fall in the range of standard curve. Therefore it may be useful to prepare several dilutions to meet this requirement: dilute samples if necessary with their respective buffer (alternatively with water).

Each buffer used in samples should be assayed alone to control eventual interference.

 

Label the tubes and record dilution factor. The dilution factor should be taken in account for the right protein concentration after interpolation from OD562nm. For example :

Sample (name)	Volume sample	Buffer (or water)	Dilution	OD@562 nm	Assayed Protein Concentration (1)	Protein Concentration in Sample (2)
#1: sample1	200	0	1	0.811	67 µg/ml	67 µg/ml
#2: sample2	20 µl	180 µl	1/10	0.592	44 µg/ml	440 µg/ml
#2: sample3

(1) Protein concentration = calculated from OD@562nm with the standard curve

(2) Protein concentration in sample = assayed protein concentration X dilution factor

 

Preparation of standards :

Uptima recommends to use the protein standard #UP36859A (BSA at 2 mg/ml) for most applications.

Prepare a fresh set of protein standards at 100 µg/ml to 0.5 µg/ml, diluted from the stock solution in the same buffer as the samples (alternatively, water may be used; check the sample buffer by analysing it versus water).

                                                                                                                .

Standard	BSA standard 2 mg/ml #UP36859A	Water or Buffer	Final protein Concentration		Figure 1 : Typical standard curve with MicroBC Assay #UP75860A
Standard A	100 µl of Stock	1900 µl	100 µg/ml
Standard B	666.6 µl of A	1000 µl	40 µg/ml
Standard C	176.5 µl of A	1000 µl	15 µg/ml
Standard D	52.6 µl of A	1000 µl	5 µg/ml
Standard E	25.6 µl of A	1000 µl	2.5 µg/ml
Standard F	10.1 µl of A	1000 µl	1 µg/ml
Standard G	5 µl of A	1000 µl	0.5 µg/ml
Blank      H	0	1000 µl	0

 

Preparation of the MicroBC Assay reagent (mix A+B+C, 25:25:1) :

Prepare the required amount of MicroBC Assay reagent by adding 25 parts of reagent A to 25 parts of reagent B, then 1 part of reagent C. Mix well (a temporary turbidity may have appeared).

 

The table below gives the needed volume for an assay with the recommended 8 points standard curve:

Tube assay (Uniplicates)*                                                               Microplate assay (Duplicates)**

Number of		Reagent A	Reagent B	Reagent C		Number of		Reagent A	Reagent B	Reagent C
standards	samples					standards	samples
8 points						8 points
	1 to 2	10 ml	10 ml	400 µl			8 to 16	5 ml	5 ml	200 µl
		20 ml	20 ml	800 µl			32 à 40	10 ml	10 ml	400 µl
		30 ml	30 ml	1,2 ml			60 to 64	15 ml	15 ml	600 µl

A duplicate (*), even a triplicate (**) analysis is recommanded for accurate determination

 

Use the mixed MicroBC Assay reagent in the next hours.

Dispose of any unused reagent because possible contamination or degradation may affect further analysis.

 

Test Tube assay:

               

	Standard protocol Working range 1µg/ml to 100µg/ml
	Allow the reagents to reach room temperature if needed
a	Pipet 1 ml of each standard, control, and sample into test tubes. Duplicates are recommended.
b	Add 1 ml of BC Assay reagent (mix A+B+C 25:25:1) per test tube, and mix
c	Incubate
	at +37°C for 60 mn
d	Cool all test tubes to room temperature and measure the optical absorbance (OD) at 562 nm against the blank (water, or buffer + MicroBC Assay reagent.
e	Plot the standard curve, and determine the protein concentration.

 

Microplate assay:

 

	Standard protocol Working range 1µg/ml to 100µg/ml
	Allow the reagents to reach room temperature if needed
a	Pipet 150µlf each standard, control, and sample into microplates wells. Duplicates or triplicates are recommended
b	Add 150µl of BC Assay reagent (mix A+B+C 25:25:1) per test well, and mix (be careful with cross-contaminations)
c	Incubate
	at +37°C for 60 mn
d	Cool the microplate at room temperature and read the optical absorbance (OD) at 562 nm against the blank (water, or buffer + MicroBC Assay reagent). Alternatively, wavelengths from 540 to 590 nm have been used.
e	Plot the standard curve, and determine the protein concentration. An example is given below in figure 1.

 

Scientific Information

Protocol :

Uptima proposes the above standard protocol, that is suitable and convenient for most applications.

Modified protocols may allow to reach different custom requirements. However, this may have incidence on the performance, the sensitivity or the interferences of substances.

-increasing the volume of sample to reagent may increase the sensitivity, but performences may be affected because of unsufficient buffering capacity, and by interfering substances.

-reading the absorbance can be done between 540 and 590 nm, for example with microplate readers lacking  a 562 nm filter. Absorbances, hence the sensitivity, is however not optimal (decreased).

Protein Standard :

Uptima complete kits includes the Bovine Serum Albumin #UP36859A because BSA is a common standard that works for most applications (see below the standard curve). Each user / application may include in the analysis other purified proteins or even any known sample (for example the extract of a reference strain). Ask Uptima for other available standards.

 

Protein to protein variations :

As with any other protein assay, but with a far lower extend, protein to protein variations may occur with some degree depending on several parameters :

. amino-acid sequences rich in cysteine, tryptophane, tyrosine may increase the MicroBC Assay reaction

. the coloured response may be affected by the primary structure (sequence order), secondary and tertiary (steric conformation) structures of the protein, isoelectric point (pI), side chains, prosthetic groups…

The MicroBC Assay can quantitate immobilized proteins (e), for example gel-coupled proteins, or cells adhering to plates.

Interfering / compatible substances

Some substances may interfere with the BC reaction. It is however remarkable that the MicroBC Assay procedure should be well known for being compatible with a lot of substances, notably most detergents (b). The following table gives some compatible and incompatible substances / concentration :

Compatible (*) Substances		Incompatible Substances
< 3% SDS		Creatinin, Cystein, Tyrosin, Tryptophan
< 2% CHAPS		> 40µM DTT and mercaptans
< 3% Tween 20		Ascorbic acid, H2O2, hydrazides
< 3% Triton X100		EGTA
< 3 M Urea		Phenol Red
< 1% DMSO, DMF		Iron, Copper salts
< 1% Glycerol
< 1 mM PMSF
< 0.5% NaN3
TBS (20 mM Tris, 150 mM NaCl, pH 7.6)
PBS (0,1 M phosphate, 150 mM NaCl, pH 7.2)
Carbonate / Bicarbonate 100 mM
Bicarbonate 40mM		Inquire for other substances

 (*) compatibility is determined if there is less than 10% variation of absorbances for the BSA standard at 40µg/ml

 

The compatible concentrations may depend on protein nature and concentration.

 

Labware may bear traces of metals that affect the BC Assay reaction. Use cleaned or disposable vials.

 

To limit the interference of some substances (b), (c), (d) :

-the samples can be diluted provided the protein concentration remains sufficient

-the interfering substance can be removed (f), for example by prior dessalting (dialysis…), precipitation (TCA…), purification…

-overcome the presence of copper chelators by increasing the A:B:C  ratio of BC Assay reagent up to 20:30:1 (v/v).

In any case, all standards, blanks and controls must be treated in the same way to preserve the accuracy of the assay.

Other information

 

ForR&D in vitro use only

 

Ask Uptima for any question

 

 

References

 

(a) Smith P et al, 1995, Measurment of protein using bicinchiconic acid, Anal. Biochem. 150, 76-85

(b) Kaushal et al, 1986, Effect of Zwitterionic buffers on measurement of small masses of protein with bicinchiconic acid, Anal.Biochem, 157, 291-294

(c) Hill et al, 1988, Protein determination using bicinchiconic acid in the presence of sulfhydryl reagents, Anal.Biochem. 170, 203-208

(d) Kessler R & Fanestil D, 1986, Interference by lipids in the determination of protein using bicinchiconic acid, Anal.biochem.159, 138-142

(e) Stich T, 1990, Determination of protein covalently bound to agarose supports using bicinchiconic acid, Anal.biochem.191. 343-346

(f) Brown et al, 1989, Protein measurement using bicinchiconic acid: elimination of interfering substances. Anal.biochem.180, 136-139

 

 

 

specification sheet N°FT75860                                                                                                                                                                                                                   revised : 9/00