National Institute of Standards & Technology

Certificate of Analysis
Standard Reference Material® 927f
Bovine Serum Albumin (7 % Solution)
(Total Protein Standard)
This Standard Reference Material (SRM) is intended primarily for use in the standardization of procedures employed
in clinical analyses for total serum protein, for critical evaluation of daily working standards used in these procedures,
and as a reference standard for assays of total protein by colorimetric methods. This SRM is a solution (mass fraction
approximately 7 %) of known protein concentration and purity. The total protein content of this SRM was determined
using the biuret reference method [1] that is recommended for use in standardizing laboratory-prepared protein
solutions and “normal” serum pools. Such standardized “normal” sera could then be used to calibrate refractometers
or other instruments for serum protein estimations. SRM 927f may also be used for other procedures, such as
gel diffusion, amino acid analysis, electrophoresis, nitrogen assays, or other tests that require well-characterized
protein for calibration or evaluation. A unit of SRM 927f consists of 10 ampoules, each containing
approximately 2.2 mL of solution. Measurements of the bovine serum albumin (BSA) concentration were made using
two independent methods including amino acid analysis and the biuret method. The results from the two approaches
are reported as follows: 1) certified BSA concentration by amino acid analysis and 2) reference BSA concentration
by the biuret method.

Certified Concentration Value: The certified concentration value for BSA as determined by amino acid analyses is
provided in Table 1. A NIST certified value is a value for which NIST has the highest confidence in its accuracy in
that all known or suspected sources of bias have been investigated or analyzed [2]. The certified value for BSA
concentration is based upon the results from isotope dilution liquid chromatography/tandem mass
spectrometry (ID-LC/MS/MS) [3].

Reference Values: The reference BSA concentration determined using the biuret method is provided in Table 2. The
biuret reference method [1] was employed to determine the BSA concentration in SRM 927f using SRM 927e as an
external standard. Reference values are non-certified values that are the best estimate of the true values based on
available data; however, the values do not meet the NIST criteria for certification and are provided with associated
uncertainties that may reflect only measurement precision, may not include all sources of uncertainty, or may reflect
a lack of sufficient statistical agreement among multiple analytical methods [2]. Reference values are provided in
Table 3 for additional properties including density and relative average molecular mass as determined using
electrospray ionization mass spectrometry.

Expiration of Certification: The certification of SRM 927f is valid, within the measurement uncertainty specified,
until 30 June 2026, provided the SRM is handled and stored in accordance with instructions given in this certificate
(see “Instructions for Storage and Use”). The certification is nullified if the SRM is damaged, contaminated, or
otherwise modified.

Maintenance of SRM Certification: NIST will monitor this SRM over the period of its certification. If substantive
technical changes occur that affect the certification before the expiration of this certificate, NIST will notify the
purchaser. Registration (see attached sheet or register online) will facilitate notification.

Overall direction and coordination of technical measurements leading to the certification were performed by
E.L. Kilpatrick of the NIST Biomolecular Measurement Division.

Statistical analysis was provided by N.F. Zhang of the NIST Statistical Engineering Division.

Michael J. Tarlov, Chief

Biomolecular Measurement Division

Gaithersburg, MD 20899 Steven J. Choquette, Director

Certificate Issue Date: 27 October 2021 Office of Reference Materials
Certificate Revision History on Last Page

SRM 927f Page 1 of 5

Acquisition of the material was performed by K.W. Phinney. Certification measurements were performed by
M.S. Lowenthal (amino acid analysis, intact molar mass) and A.B. Green (biuret analysis, density) of the NIST
Biomolecular Measurement Division.

Support aspects involved in the issuance of this SRM were coordinated through the NIST Office of Reference
Materials.

NOTICE AND WARNINGS TO USERS

Warning: SRM 927f IS INTENDED FOR RESEARCH USE. The blood used in the preparation of
SRM 927f Bovine Serum Albumin (7 % Solution) was collected from cattle sourced in the United States. Only blood
from cattle/carcasses that have passed ante-mortem and post-mortem U.S. Department of Agriculture (USDA) Food
Safety Inspection Service (FSIS) inspection was used. This material has not come from cattle that have been in a herd
in which a case of Bovine Spongiform Encephalopathy (BSE) has appeared, and the product does not contain, and is
not derived from, specified risk material as defined in Commission Decision 97/534/EC. No bovine blood was used
from tuberculosis and/or brucellosis reactors. No bovine blood was used from animals subject to emergency slaughter
or identified as U.S. Suspect. There were no additives to the pooled serum prior to protein purification.

INSTRUCTIONS FOR STORAGE AND USE

Storage: This SRM is supplied to the user in sealed ampoules. The SRM should be stored in a refrigerator at a
temperature between 2 °C and 8 °C. The ampoules should not be frozen because of possible breakage of ampoules
during the thawing process.

Instructions for Use: Once an ampoule is opened, the solution should be used promptly. Any unused solution in
opened ampoules should be discarded.

Preparation of Dilutions: Protein solutions of lower concentration may be prepared by transferring the appropriate
aliquot to a volumetric flask and diluting to volume. Diluents are not furnished with the SRM; an aqueous
sodium chloride diluent, such as a solution having a concentration of 0.15 mol/L, may be used. Caution should be
taken when performing dilutions greater than 100-fold due to the potential for protein loss (possibly due to protein
absorption on laboratory equipment).

Inappropriate Uses: This SRM is not intended to be used as a standard for dye-binding tests as the dye-binding
characteristics of BSA will likely be dissimilar to other proteins being assayed. Additionally, this SRM is not intended
for checking pre-calibrated refractometers, for immunochemical methods, or as an additive for bilirubin
standardization.

SOURCE, PREPARATION, AND ANALYSIS(1)

Source and Preparation: SRM 927f was prepared and packaged by Equitech-Bio, Inc. (Kerrville, TX). The source
bovine serum was produced in the U.S. at establishments registered with the USDA and is intended only for
manufacture into products for use in in vitro diagnostic, research, and further manufacturing or technical purposes.
The BSA for this SRM was diluted from 30 % BSA commercial stock (Equitech-Bio, Inc., Kerrville, TX) to
approximately 7 % using 0.02 mol/L sodium chloride, and the pH adjusted to 6.5 to 6.8 with sodium hydroxide. The
material was sterilized by membrane filtration, placed into sterile glass argon-flushed ampoules and flame sealed.

Analysis: All analyses in the value assignment of SRM 927f were performed at NIST.

Measurement of BSA Concentration by Amino Acid Analysis (ID-LC/MS/MS): The amino acid analysis method
involved isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) [3]. Underivatized
samples of SRM 927f and SRM 927e (as a control) were diluted and then combined with stable isotope-labeled
analogs of phenylalanine, proline, isoleucine, leucine, and valine followed by hydrolysis with liquid-phase
hydrochloric acid (HCl) for 48 h at approximately 130 ºC in a sealed pressure vessel. After hydrolysis, the samples
were lyophilized and then reconstituted with 0.1 mL/L formic acid in water. Amino acids were separated using
gradient-elution mixed-mode chromatography on a reversed-phase analytical column with embedded
acidic ion-pairing groups. Measurements were performed on a triple quadrupole mass spectrometer, monitoring
specific transitions for each amino acid. The measurements were calibrated using solutions prepared from

(1)Certain commercial equipment, instruments, or materials are identified in this certificate to adequately specify the

experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards
and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

SRM 927f Page 2 of 5

commercially available amino acid certified reference materials with known purity and uncertainty. Data were
collected for phenylalanine, proline, isoleucine, leucine and valine. Based upon the known amino acid sequence for
BSA, the mass fraction of BSA was calculated from the mass fractions determined for each of the amino acids using
the appropriate molar masses and molar stoichiometries. BSA concentration was determined as the product of the
BSA mass fraction multiplied by the density of SRM 927f (described below) with correction to a gram per liter basis.

Measurement of BSA Concentration (Biuret): The reference BSA concentration was measured using the biuret
reference method for total serum protein [1]. The measurements involve a direct comparison between the current
SRM 927f and previously issued SRM 927e and were performed by spectrophotometry.

Additional Analyses: Density measurement was performed gravimetrically [4]. Relative average molecular mass
was determined using liquid chromatography/mass spectrometry (LC/MS). Measurements were performed on a
time-of-flight mass spectrometer operated in the positive ion mode via LC using a commercial C18 column and adding
0.1 mL/L trifluoroacetic acid as an ion-pairing agent. Reversed phase gradient elution was performed by increasing
the mobile phase acetonitrile content from 200 mL/L to 950 mL/L (balance water). The relative average molecular
masses of the four major forms of BSA found in SRM 927f are shown in Table 3 in decreasing order of abundance.
The previous issues of this material, SRM 927e and SRM 927d, had a similar range of molecular masses.

Homogeneity Analysis: The homogeneity assessment was made at the time the certification analyses were
performed. A stratified sampling plan was devised to test for homogeneity across the lot of ampoules. There was no
apparent trend in the data when plotted against the sequence in which the ampoules were prepared.

Certified Value: The measurand is the total concentration of bovine serum albumin. Metrological traceability is to
the International System of Units (SI) derived unit for mass concentration (expressed as gram per liter). The
uncertainty provided with the measured BSA certified concentration value is an expanded uncertainty about the mean
to cover the measurand with approximately 95 % confidence, consistent with the ISO/JCGM Guide [5]. The expanded
uncertainty is calculated as U = kuc, where uc is the combined uncertainty, and k is a coverage factor corresponding to
approximately 95 % confidence for this analyte [5]. For the certified value shown below, k = 2.

Table 1. Certified Bovine Serum Albumin Concentration by Amino Acid Analysis

BSA Concentration: 83.2 g/L ± 0.7 g/L

Reference Value: The reference value in Table 2 is based specifically on the biuret reference method. The measurand
is the bovine serum albumin concentration as determined using the biuret method. Metrological traceability is to the
SI derived units for mass concentration (expressed as gram per liter). The uncertainty provided with the reference
value in Table 2 is an expanded uncertainty about the mean to cover the measurand with approximately
95 % confidence; it incorporates Type B uncertainty components related to the analyses, consistent with the
ISO/JCGM Guide and with its Supplement 1 [5,6]. The expanded uncertainty is calculated as U = kuc, where uc is the
combined uncertainty and k is a coverage factor corresponding to approximately 95 % confidence for this analyte [5].
For the reference value shown in Table 2, k = 2.

Table 2. Reference Bovine Serum Albumin Concentration by the Biuret Method

BSA Concentration: 87.6 g/L ± 2.7 g/L

SRM 927f Page 3 of 5

Additional Reference Values: The reference values are based on the method used for each measurand as described
above. Metrological traceability of the density value is to the SI units for gram per milliliter. The uncertainty provided
with each reference value in Table 3 is an expanded uncertainty about the mean to cover the measurand with
approximately 95 % confidence. The expanded uncertainty is calculated as U = kuc, where uc is the combined
uncertainty and k is a coverage factor corresponding to approximately 95 % confidence for each analyte [6]. For the
reference values shown in Table 3, k = 2.

Table 3. Additional Reference Values for Properties of SRM 927f

Density(a)
(g/mL)

1.02181 ± 0.0005

Relative Average Molecular Mass of the Major Molecular Forms of BSA(b)

(unitless)

66 432
66 453
66 550
66 597
(a) Theuncertainty of the solution density was combined with the uncertainty of the amino acid analysis in determining the expanded
uncertainty of the BSA concentration.
(b) Decreasing order of abundance.

Additional Information: The theoretical relative average molecular mass of BSA was calculated to be 66 398.1.
The calculation of the average relative molecular mass of BSA is based on the reported amino acid sequence [7].

10 20 25 30 40 50 60
MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF
70 80 90 100 110 120
DEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP
130 140 150 160 170 180
ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY
190 200 210 220 230 240
ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA
250 260 270 280 290 300
RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE
310 320 330 340 350 360
CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR
370 380 390 400 410 420
HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK
430 440 450 460 470 480
LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL
490 500 510 520 530 540
NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP
550 560 570 580 590 600
DTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV
607
STQTALA

The sequence of the mature protein is not expected to include the signal peptide which is removed as part of normal
post-translational processing. Therefore, the sequence of circulating BSA includes only amino acids from position
25 to 607 as indicated by the non-underlined text above. BSA is also reported to contain 17 disulfide bonds [7] at the
following cysteine residue pairs: 77-86, 99-115, 114-125, 147-192, 191-200, 223-269, 268-276, 288-302, 301-312,
339-384, 383-392, 415-461, 460-471, 484-500, 499-510, 537-582, and 581-590.

SRM 927f Page 4 of 5

 REFERENCES

[1] Doumas, B.T.; Bayse, D.D.; Carter, R.J.; Peters, T., Jr.; Schaffer, R.; A Candidate Reference Method for
Determination of Total Protein in Serum. I. Development and Validation; Clin. Chem., Vol. 27, pp. 1642–1650
(1981).
[2] Beauchamp, C.R.; Camara, J.E.; Carney, J.; Choquette, S.J.; Cole, K.D.; DeRose, P.C.; Duewer, D.L.;
Epstein, M.S.; Kline, M.C.; Lippa, K.A.; Lucon, E.; Molloy, J.; Nelson, M.A.; Phinney, K.W.; Polakoski, M.;
Possolo, A.; Sander, L.C.; Schiel, J.E.; Sharpless, K.E.; Toman, B.; Winchester, M.R.; Windover, D.;
Metrological Tools for the Reference Materials and Reference Instruments of the NIST Material Measurement
Laboratory; NIST Special Publication (NIST SP) 260-136, 2021 edition; U.S. Government Printing Office:
Washington, DC (2021); available at https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP.260-136-
2021.pdf (accessed Oct 2021)
[3] Bunk D.M.; Lowenthal M.S; Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry for
Quantitative Amino Acid Analysis; In Amino Acid Analysis. Methods and Protocols; part of the Methods
in Molecular Biology book series, Vol 2030; Alterman, M.A., Ed.; Humana: New York, NY, pp. 143‒151
(2019).
[4] Sniegoski, L.T.; Moody, J.R.; Determination of Serum and Blood Densities; Anal. Chem., Vol 51, pp. 1577–1578
(1979).
[5] JCGM 100:2008; Evaluation of Measurement Data — Guide to the Expression of Uncertainty in Measurement
(GUM 1995 with Minor Corrections); Joint Committee for Guides in Metrology (JCGM) (2008); available at
https://www.bipm.org/en/publications/guides (accessed Oct 2021); see also Taylor, B.N.; Kuyatt, C.E.;
Guidelines for Evaluating and Expressing the Uncertainty of NIST Measurement Results; NIST Technical
Note 1297; U.S. Government Printing Office: Washington, DC (1994); available at
https://www.nist.gov/pml/nist-technical-note-1297 (accessed Oct 2021).
[6] JCGM 101:2008; Evaluation of Measurement Data — Supplement 1 to the “Guide to the Expression of
Uncertainty in Measurement” — Propagation of Distributions Using a Monte Carlo Method; JCGM (2008);
available at https://www.bipm.org/en/publications/guides (accessed Oct 2021).
[7] The UniProt Consortium; UniProt Database (2021); available at https://www.uniprot.org (accessed Oct 2021).

Certificate Revision History: 27 October 2021 (Amended uncertainty value for biuret protein concentration; editorial changes);
23 June 2021 (Original certificate date).

Users of this SRM should ensure that the Certificate of Analysis in their possession is current. This can be
accomplished by contacting the SRM Program: telephone (301) 975-2200; e-mail [email protected]; or via the
Internet at https://www.nist.gov/srm.

SRM 927f Page 5 of 5