Bicinchoninic Acid Protein Assay Kit
Product Code
BCA1 AND B 9643
TECHNICAL BULLETIN
Synonym: BCA
Product Description
Protein determination is one of the most commonoperations performed in biochemical research. Theprinciple of the bicinchoninic acid (BCA) assay issimilar to the Lowry procedure,
1
in that both rely on theformation of a Cu
2+
-protein complex under alkalineconditions, followed by reduction of the Cu
2+
to Cu
1+
.The amount of reduction is proportional to the proteinpresent. It has been shown that cysteine, cystine,tryptophan, tyrosine, and the peptide bond
2
are able toreduce Cu
2+
to Cu
1+
.
BCA forms a purple-bluecomplex with Cu
1+
in alkaline environments, thusproviding a basis to monitor the reduction of alkalineCu
2+
by proteins.
3
The BCA assay is more sensitive and applicable thaneither biuret or Lowry procedures. In addition, it hasless variability than the Bradford assay. The BCAassay has many advantages over other proteindetermination techniques:
•
It is easy to use.
•
The color complex is stable.
•
There is less susceptibility to detergents.
•
It is applicable over a broad range of proteinconcentrations.In addition to protein determination in solution, theBCA protein assay has other applications, includingdetermination of protein covalently bound to agarosesupports and protein adsorbed to multiwell plates.There are two distinct ways to perform a proteinassay. A protein assay can be set up to measure theconcentration of the unknown protein sample (mg/ml),or it can be set up to determine the total amount ofprotein in the unknown protein sample (mg). The BCAassay has a linear concentration range between200-1,000
µ
g of protein per milliliter. In the standardassay, only 0.1 ml protein sample is used, so theassay has a total linear protein range of 20-100
µ
g.
Reagents Provided
Bicinchoninic Acid Solution (B9643)Reagent A is a 1,000 ml solution containingbicinchoninic acid, sodium carbonate, sodium tartrate,and sodium bicarbonate in 0.1 N NaOH(final pH 11.25).Copper(II) Sulfate Pentahydrate 4% Solution (C2284)Reagent B is a 25 ml solution containing4% (w/v) copper(II) sulfate pentahydrate.Protein Standard (Bovine Serum Albumin - BSA)Solution (P0914)This product is supplied in 5 flame-sealed glassampules, each containing 1.0 ml of a solutionconsisting of 1.0 mg/ml bovine serum albumin in0.15 M NaCl with 0.05% sodium azide as apreservative.
Materials required depending on assay formatused but not provided
•
Spectrophotometer capable of accuratelymeasuring absorbance in the 560 nm region.
•
96 well plates, Product Code M0156
•
96 well plate sealing film, Product Code Z369667
•
Test tubes, 13 x 100 mm, Product Code Z144371
•
1 ml Disposable Plastic Cuvets,Product Code C5416
Precautions and Disclaimer
This product is for R&D use only, not for drug,household, or other uses. Please consult the MaterialSafety Data Sheet for information regarding hazardsand safe handling practices.
Preparation Instructions
The BCA Working Reagent is prepared by mixing50 parts of Reagent A with 1 part of Reagent B. Mixthe BCA Working Reagent until it is light green incolor.
Storage/Stability
Store Reagents A and B at room temperature.Reagent A, without Reagent B added, is stable for atleast one year at room temperature in a closedcontainer. The BCA Working Reagent (Reagent Amixed with Reagent B) is stable for one day.Store the Protein Standard at 2–8
°
C.
Procedures
In the standard assay, 20 parts of the BCA WorkingReagent are then mixed with 1 part of a proteinsample. For the 96 well plate assay, 8 parts of theBCA Working Reagent are mixed with 1 part of aprotein sample. The sample is either a blank, a BSAprotein standard, or an unknown sample. The blankconsists of buffer with no protein. The BSA proteinstandard consists of a known concentration of bovineserum albumin, and the unknown sample is thesolution to be assayed.BCA assays are routinely performed at 37
°
C.Color development begins immediately and can beaccelerated by incubation at higher temperatures.Higher temperatures and/or longer incubation timescan be used for increased sensitivity. Incubation atlower temperatures can slow down color development(see Procedures A and B). The absorbance at 562nm is recorded and the protein concentration isdetermined by comparison to a standard curve.
A. Standard 2.1 ml Assay Protocol
(Linear concentration range is 200-1,000
µ
g/ml or20-100
µ
g of total protein)This is the standard assay that can be performed in atest tube. This procedure uses 0.1 ml of a proteinsample and 2 ml of the prepared BCA WorkingReagent. The instructions are a step-by-stepprocedure on how to perform the standard assay. If anonstandard assay is used (96 well plate) adjust thevolumes accordingly.Note
:
It is necessary to create a standard curve duringeach assay, regardless of the format used.1. Prepare the required amount of BCA WorkingReagent needed for the assays (Table 1). Thefinal volume used in the assay depends upon theapplication and the equipment available. Table 1can be used to determine the volume of BCAWorking Reagent to prepare, depending on howmany blanks, BSA protein standards, andunknown samples are to be assayed. Combinethe volumes of Reagents A and B specified in thetable. Mix until the BCA Working Reagent is auniform, light green color.
Table 1.
Volume of BCA Working Reagent to prepare. This isdependent on how many blanks, BSA proteinstandards, and unknown samples are to be assayed.Number of Assays Amount of Each Reagent UsedNumberof 2.1 mlStandardTest tubeassaysNumber ofwells in a96 wellplate assayReagentA(ml)ReagentB(ml)Totalvolume ofBCAWorkingReagent(ml)4 40 8 0.16 8.168 80 16 0.32 16.329 96 19 0.38 19.3812 127 25 0.5 25.52. Prepare standards of different concentrations.These BSA protein standards can range from200-1,000
µ
g/ml (20-100
µ
g of total protein). Thisis accomplished by making serial dilutions startingfrom the 1 mg/ml standard, and then using 0.1 mlof each diluted standard in the assay. It is best tomake the dilutions in the same buffer as theunknown sample (Table 2). Deionized water maybe used as a substitute for the buffer, but anyinterference due to the buffer will not becompensated for in the BSA protein standards.
Table 2
EXAMPLE of Standard Assay Set Up TableFor protein samples with unknown concentrations, itmay be necessary to prepare a dilution scheme toensure the concentration is within the linear range of200-1,000
µ
g/ml. Two different unknown samples arerepresented in Table 2 by tubes 7 and 8. Tube 7 is anunknown sample with a 5-fold dilution, while tube 8 isa different unknown sample at a 10-fold dilution.Researchers must determine their own dilutionschemes based on their estimation of theconcentration of each unknown sample.TubeNo.Sample(ml)[BSA]Protein Standard(
µ
g/ml)BCAWorkingReagent(ml)1 0.1 0 22 0.1 200 23 0.1 400 24 0.1 600 25 0.1 800 26 0.1 1,000 27 0.1 (unknown 1) 28 0.1 (unknown 2) 23. Add 2 ml of the BCA Working Reagent to 0.1 ml ofeach BSA protein standard, blank, and unknownsample. Vortex gently for thorough mixing. Thetotal liquid volume in the test tube is 2.1 ml.4. The following incubation parameters may be used:60
°
C for 15 minutes
Or
37
°
C for 30 minutes
Or
25
°
C (Room Temperature) from 2 hours toovernight5. If required, allow the tubes to cool to roomtemperature.6. Transfer the reaction solutions into a cuvet.7. Measure the absorbance of the solution at562 nm. Color development continues slowly aftercooling to room temperature, but no significanterror is seen if all the tubes are read within10 minutes of each other. Create an assay tableas needed and a standard curve based on eitherthe BSA protein standard concentration or on theamount of protein present in the BSA proteinstandard (Examples are shown in the resultsbelow).8. Determine protein concentration by comparison ofthe absorbance of the unknown samples to thestandard curve prepared using the BSA proteinstandards.Results Based on the Standard AssayCreate a table with the absorbance results obtainedduring the assay. A separate standard curve shouldbe generated for each assay performed. The amountof protein for tubes 1-6 was obtained from the knownamount of BSA protein standard added
.
Note: The data below should not be used as areplacement of a standard curve. The absorbance ofthe BSA protein standards (tubes 1-6) in each assaywill differ from those presented here. The amount ofprotein recorded for tubes 7 and 8 was obtained fromthe standard curve.
Table 3.
EXAMPLE of Assay Data TableTubeNo.A
562
NetA
562
Amount ofprotein (
µ
g)in sample[Protein]of proteinsample(
µ
g/ml)DilutionFactor1 0.045 0 0 0 -2 0.207 0.162 20 200 -3 0.364 0.319 40 400 -4 0.510 0.465 60 600 -5 0.661 0.616 80 800 -6 0.823 0.778 100 1,000 -7 0.587 0.542 70 700 58 0.743 0.698 90 900 10After obtaining the results, create a standard curve todetermine the protein concentration in the unknownsample. Plot the Net Absorbance at 562 nm versusthe BSA protein standard concentrations(
µ
g/ml, Tubes 1-6).
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