Albumin From Bovine Serum

Description

Bovine serum albumin is a protein which has a key role in regulating blood colloidal osmotic pressure. A water-soluble protein composed of 583 amino acids, BSA has a calculated molecular weight of 66,430 Daltons. Six α-helices form three homologous domains of BSA. Depending on pH, BSA undergoes reversible conformational isomerization. The native structure of the protein becomes reactive and flexible on heating.
This BSA product is specifically evaluated and tested for the following properties:
Very low protease content / protease-free
Very low fatty acid content / fatty acid-free
Protease-free BSA is especially useful for applications like:
Enzyme assays
Protein-based assays
Protease-sensitive techniques that include:
a. Enzyme immunoassay (EIA)
b. Nucleic acid hybridization
c. Radioimmunoassay (RIA)

Description

Acetylated BSA may be used as a positive control for ELISA or WB when employing an acetyl-lysine monoclonal or polyclonal antibody. Cayman’s Acetyl Lysine Monoclonal Antibody (Clone 7F8) can detect less than 1 ng of this positive control by immunoblotting. Dilutions from a 1 mg/ml stock solution and 2X Laemmli Buffer may be prepared for WB use. For example, dissolve the 1 mg lyophilized protein in 1 ml of purified water (aliquot and freeze for later, use as needed), then make ten-fold dilutions until the desired concentration range is obtained. Finally dilute the product 1:1 in 2X Laemmli Buffer. Incubate the prepared sample for five minutes in boiling water followed by five minutes on ice prior to loading gels.Histone subunit modifications such as lysine acetylation are regulated by the activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Epigenetic modifications such as protein acetylation directly influence cellular genetic programs including those contributing to cancer cell viability.

Chemical Properties

Crystalline powder

Uses

Suitable for cell culture media supplement.
Bovine serum albumin (BSA) Fraction V has uses in many applications such as stabilizing proteins in solution, blocking non-specific binding in immuno-assays, and as a protein standard in various protein assays.
Stabilizer for proteins and enzymes in solution
Blocking agent for immuno-assays (ie, western blot, IHC, IF)
Minimizes non-specific antibody binding
IgG- and protease-free
50 g of solid powder
Bovine serum albumin (BSA) is commonly used as a stabilizing agent for proteins and enzymes, including dilute solutions of antibody. It is also used as a blocking agent to reduce non-specific antibody binding in immuno-detection procedures such as western blotting, immunofluorescence, and IHC. It is also suitable for use in protease sensitive assays such as RIA and EIA, as well as nucleic acid hybridization. This BSA is IgG- and protease-free.

Uses

Select the right albumin for your application with the Albumin Selection Guide

BSA has various uses in molecular biology applications, such as:

• As an added component in solutions of enzymes or of other proteins, to prevent adhesion of the enzyme or other protein of interest to the reaction tube surface or to pipette surfaces
• As an inert enzyme or protein stabilizer during incubation, to allow the enzyme or protein of interest to function properly

Uses

Protease-sensitive Immunoassays such as RIA, EIA, Fluorescent and Chemiluminescent, Protein Standard, Diluent, Protein, Conjugate or Enzyme Stabilizer. Bovine Serum Albumin is applicable for numerous biochemical applications including ELISAs (Enzyme-Linked Immunosorbent Assay), immunoblots, and immunohistochemistry. Frequently used as a blocking reagent; particularly useful with casein-sensitive antibodies, such as phospho-specific antibodies. Also used as a nutrient in cell and microbial culture. In restriction digests, BSA is used to stabilize some enzymes during digestion of DNA and to prevent adhesion of the enzyme to reaction tubes and other vessels. Commonly used to determine the quantity of other proteins, by comparing an unknown quantity of protein to known amounts of BSA. Hybridization, Selected Cell Culture applications

Definition

The word bovine means “cow,” and bovine serum albumin (BSA) is a protein that comes from cows. In particular, BSA is a type of protein called albumin, which is found in large amounts in the blood of cows. Blood is a mixture of red blood cells, which is why blood looks red, and many different types of proteins that are dissolved in water. If the cells in blood were removed, you would be left with a clear liquid, called serum. Thus, BSA is the albumin protein that is found in the blood of cows. In the blood, the job of albumin is to carry other proteins from place to place.

Application

Bovine Serum Albumin has been used:
as a constituent of medium used for the differentiation of human pluripotent stem cells
in enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA
to permeabilize fetal cortical cells
This Sigma A7030 BSA has specifically been used as a component of blocking buffer in a peptide-based ELISA assay for analysis of COVID-19 / SARS-CoV-2 samples (Poh, C.K. et al., Nat. Commun., 11(1), 2806 (2020)).

General Description

Bovine serum albumin (BSA) has an important role in the regulation of blood colloidal osmotic pressure. BSA is the major protein in bovine serum, and is composed of 583 amino acids with a calculated molecular weight of 66,430 Daltons. BSA is used in many different applications and fields, such as:

• Protein quantitation (as a standard)
• Immunochemistry (as a blocking agent)
• Cell culture (as a nutrient)

• Enzyme assays
• Protein-based assays
• Protease-sensitive techniques that include:

Biological Activity

Bovine Serum Albumin is a BSA (bovine serum albumin) is a widely used blocking reagent for use in immunohistochemistry (IHC), Immunocytochemistry (ICC), ELISAs and Western blotting. Used to block non-specific binding of antibodies. Can also be used as a protein concentration standard in Bradford assay for protein quantification. BSA is also used in cell culture.

Biochem/physiol Actions

Certain conformational and primary-sequence epitopes of BSA are suspected allergens in human beef and milk allergies.

storage

Store at +4°C

Purification Methods

Albumin is purified by dissolving it in conductivity water and passage at 2-4o through two ion-exchange columns, each containing a 2:1 mixture of anionic and cationic resins (Amberlite IR-120, H-form, Amberlite IRA-400, OH -form). This treatment removes ions and lipid impurities. Care is taken to exclude CO2, and the solution is stored at -15o. [M.ller et al. Trans Faraday Soc 57 312 1961.] More complete lipid removal is achieved by lyophilising the de-ionised solution, covering the dried albumin (human serum) with a mixture of 5% glacial acetic acid (v/v) in iso-octane (previously dried with Na2SO4) and allowing it to stand at 0o (without agitation) for upwards of 6hours before decanting and discarding the extraction mixture, washing with iso-octane, re-extracting, and finally washing twice with iso-octane. The purified albumin is dried under vacuum for several hours, then dialyzed against water for 12-24hours at room temperature, lyophilised, and stored at -10oC [Goodman Science 125 1296 1957]. It has been recrystallised in high (35%) and in low (22%) EtOH solutions from Cohn's Fraction V. The high EtOH recrystallisation is as follows: To 1kg of Fraction V albumin paste at -5o is added 300mL of 0.4 M pH (pH 5.5) acetate buffer in 35% EtOH pre-cooled to -10o and 430 mL of 0.1 M NaOAc in 25% EtOH also at -10o. Best results are obtained by adding all of the buffer and about half of the NaOAc and stirring slowly for 1hour. The rest of the NaOAc is added when all the lumps have disintegrated. The mixture is set aside at -5o for several days to crystallise. 35% EtOH (1 L) is then added to dilute the crystalline suspension and lower the ionic strength prior to centrifugation at -5o (yield 80%). The crystals are further dissolved in 1.5 volumes of 15% EtOH/0.02M NaCl at -5o and clarified by filtration through washed, calcined diatomaceous earth. This solution may be recrystallised by re-adjusting to the conditions in the first crystallisation, or it may be recrystallised at 22% EtOH with the aid of a very small amount of decanol (enough to give a final concentration of 0.02%). Note that crystallisation from lower EtOH concentration gave better purification (i.e. by removing globulins and carbohydrates) and producing a more stable product. The low EtOH recrystallisation is as follows: To 1kg of Fraction V at -10o to -15o is added 500mL of 15% EtOH at -5o, stirred slowly until a uniform suspension is formed. To the 15% EtOH (500mL) is added sufficient 0.2M NaHCO3 solution (125-150mL) at 0o to bring the pH (1:10 dilution) to 5.3. Some temperature rise occurs, and care must be taken to keep the temperature < -5o. If the albumin is incompletely dissolved a small amount of H2O is added (100mL at a time at 0o, allowing 15minutes between additions). Undissolved albumin can be easily distinguished from small amounts of undissolved globulins, or as the last albumin dissolves, the appearance of the solution changes from milky white to hazy grey-green in colour. Keep the solution at -5o for 12hours and filter by suspending in 15g of washed fine calcined diatomaceous earth, and filtering using a Büchner funnel precoated with coarser diatomaceous earth. The filtrate may require two or more similar filtrations to give a clear solution. To crystallise the filtrate, add through a capillary pipette, and with careful stirring, 1/100volume of a solution containing10% decanol and 60% EtOH (at -10o), and seed with the needle-type albumin crystals. After 2-3days, crystallisation is complete. The crystals are centrifuged off. These are suspended with gentle mechanical stirring in one-third their weight of 0.005 M NaCl pre-cooled to 0o. With careful stirring, H2O (at 0o) is added slowly in an amount equal to 1.7 times the weight of the crystals. At this stage there is about 7% EtOH, and the temperature cannot be made lower than -2.5o to -1o. Clarify, and collect as above. [Cohn et al. J Am Chem Soc 69 1753 1947.] Human serum albumin has been purified similarly with 25% EtOH and 0.2% decanol. The isoelectric points of bovine and human serum albumins are 5.1 and 4.9, respectively.

References

[1] LAEMMLI U K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4[J]. Nature, 1970, 227 5259: 680-685. DOI: 10.1038/227680a0
[2] LOIDL P. Histone acetylation: facts and questions.[J]. Chromosoma, 1994, 103 7: 441-449. DOI: 10.1007/bf00337382
[3] PAUL A MARKS  Ronald B. Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug[J]. Nature biotechnology, 2007, 25 1: 84-90. DOI: 10.1038/nbt1272
[4] JESSICA E. BOLDEN  Ricky W J  Melissa J Peart. Anticancer activities of histone deacetylase inhibitors[J]. Nature Reviews. Drug Discovery, 2006, 5 9: 769-784. DOI: 10.1038/nrd2133