Streptavidin from Streptomyces avidinii 链霉亲合素 (≥13u/mg)

Product Description

Appearance: lyophilized white powder E1% = 34 at 280 nm (solvent: 0.1 M ammonium perchlorate, pH 8.0)

Structure: References have cited the molecular weight for the native protein as 60,000; it contains four subunits of 15,000.1,2,3 The molecular weight of affinitypurified streptavidin was reported more recently as 75,000; under extreme denaturing conditions, giving a major band at about 18,000.4 However, the structure of “truncated streptavidin” is also discussed by this author.4 Isoelectric point: 6.45, 5-66

Additional analytic methods and data have been reported.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

This protein is isolated by a proprietary method developed by Sigma. The affinity purification step uses iminobiotin-agarose, Sigma product I4507, binding at pH 9 and eluting at pH 2. Extensive dialysis against distilled water makes this product "essentially saltfree.” 9 A similar affinity preparation has been reported.4 Due to affinity purification, the product is active, but appears to be heterogeneous on an SDS PAGE gel, giving a number of bands from 12,000 to 18,000.

Storage/Stability

The dry solid is stable at least three years stored frozen.

Solubility / Solution Solubility

Streptavidin is soluble in water at 1 mg/mL, or in phosphate buffered saline (PBS). Solutions in PBS or water can be stored several months at 4°C or for more than a year as frozen aliquots.

Product Profile

Streptavidin is a protein produced by the bacterium Streptomyces avidinii. It has four binding sites for biotin, as does avidin. Both have been used extensively as probes in immunochemical systems, conjugated to antibodies, enzymes or fluorochromes. However, avidin is highly basic (pI ~ 10.5), compared to streptavidin (pI ~ 5-6). Avidin has a slightly higher binding capacity for biotin than streptavidin does, but it has glycosylated side chains which give higher backgrounds in blotting techniques than does streptavidin, which is not a glycoprotein. Streptavidin is more resistant than avidin to dissociation into subunits by guanidinium chloride.11

This product is assayed for biotin binding by a modification of Green’s method.12 (The procedure is given below.)

In blotting techniques, blocking agents are frequently used to reduce non-specific binding. Gelatin, albumin and casein (as nonfat dried milk) are commonly used for this purpose. However, it is advised that the use of nonfat dried milk be avoided if possible; Hoffmann et al. report that nonfat dried milk contains an "inhibitor of the biotin-streptavidin interaction and it is recommended that milk be dialyzed or used at lower concentrations when it is employed as a diluent of streptavidin."13

Biotin Binding Assay

This assay is modified from Green’s spectrophotometric assay for the determination of avidin.12

REAGENTS:

A. 0.1 M sodium phosphate buffer pH 7.0

B. 2 mM Biotin (Dissolve 4.9 mg B4501 in 10 mL buffer A)

C. 10 mM 2-(4'-Hydroxyazobenzene)-benzoic acid [HABA] (Dissolve 24.2 mg H5126 in 10.0 mL buffer A.)

D. Streptavidin (Dissolve approx. 0.1 mg per mL buffer A.)

E. Avidin control. (Dissolve Avidin A9275 at 0.1 mg per mL buffer A.)

PROCEDURE:

To a 3.0 mL cuvette (1.0 cm light path), add 2.0 mL streptavidin solution or avidin control.

Zero the spectrophotometer using this solution at 500 nm at 25°C.

Add 0.05 mL HABA solution (Reagent C). Mix and record A500.

Add 0.05 mL Biotin solution (Reagent B). Mix and record A500.

Calculate )A500. Note that total volume in cuvette = 2.10 mL.

CALCULATIONS:

Streptavidin (mg/mL of original solution) = 2.1 x 15 x )A500 = 0.65 )A500

2 x 24

2.1 = total volume in cuvette

2 = sample volume

15 = milliequivalent weight of streptavidin for biotin binding

24 = millimolar extinction coefficient of streptavidin-

HABA complex at pH 7.0

Note: This is the protocol currently used at Sigma to determine biotin binding, although this is not in a standardized format.

Sigma’s Immunochemistry group also offers Extravidin™, a modified avidin that has the advantage of binding specificity of avidin along with the reduced background seen with streptavidin. See Sigma’s catalog listings for a large number of conjugate products prepared from Extravidin™.