STREPTAVIDIN-PLUS™ - ProZyme

STREPTAVIDIN-PLUSSPECIFICATIONSProduct Code: SA20Specific activity: 14.0 U/mgContains 0.9 mg protein/mglyophilizate (balance is sodiumchloride).Shipped with ice pack for next daydelivery. Store at -20C.Stability: Streptavidin-plus is stableas a lyophilizate for at least one yearwhen stored desiccated at -20C.ProZyme Streptavidin-plus is manufacturedby a proprietary process and has somewhatdifferent properties from ProZyme’s SA10Streptavidin. In particular, passiveadsorption to polystyrene is enhanced, andthermal stability of the protein may beimproved. Streptavidin-plus retains the highspecific activity of ProZyme’s SA10Streptavidin.Extinction Coefficient: E = 32 (estimate)Origin: USAASSAYThe biotin-binding activity of streptavidin isdetermined using a modification of the dyebindingassay of Green (1970). One unit willbind one microgram of d-biotin at pH 7.0.Reagents10 mM 2-(4'-hydroxyazobenzene)benzoicacid (Sigma) dissolved in 10 mM sodiumhydroxide (HABA)0.2 M sodium phosphate, pH 7.02 m M d-biotin in 0.1 M sodium phosphate,pH 7.0Streptavidin sample - dissolve at 5-10 mg/mlin de-ionized water.ProcedureAdjust spectrophotometer to read at 500 nm.To two tubes labeled A and B add as follows:Streptavidin-plussampleAB0.05 ml 0.05 mlPhosphate Buffer 0.5 ml 0.5 mlHABA stock 0.1 ml 0.1 mlBiotin stock -- 0.25 mlH2O 0.35 ml 0.1 mlTotal Volume 1.0 ml 1.0 mlAfter mixing, zero the spectrophotometerwith water and read the absorbances intubes A and B.These suggestions and data are based on information we believe to be reliable. They are offered in good faith, but without guarantee, as conditions and methods of use of our products are beyond ourcontrol. We recommend that the prospective user determine the suitability of our materials and suggestions before adopting them on a commercial scale.Suggestions for use of our products or the inclusion of descriptive material from patents and the citation of specific patents in this publication should not be understood as recommending the use of ourproducts in violation of any patent or as permission to license to use any patents of ProZyme, Inc.

Calculationwhere:M = formula weight of d-biotin (244 g/mole)V = volume of assay in liters (.001 liters)v = volume of streptavidin sample inmilliliters (.05 ml as written)C = concentration of streptavidin in sample(mg/ml) = net molar extinction coefficient ofHABA-streptavidin complex at 500 nm-1(34,500 M )The measured specific activity of a given lotdepends on the assay method used, as wellas the method used to quantitate theStreptavidin (gravimetric vs spectrophotometric).For comparison purposes, ProZymealso reports the results of a biotin titrationassay.Biotin-binding capacity:Dilute the 1 mg/ml biotin solution to~0.1 mg/ml in Ammonium CarbonateBuffer. Prepare at least 1 ml.Add 1 ml of 0.1 mg/ml Streptavidin sample tocuvette. Take initial reading of unboundstreptavidin.Add diluted biotin (0.1 mg/ml) in incrementsof 3 l. Mix carefully after each additionand take absorbance readings.Continue adding biotin until absorbancereadings plateau or decline repeatedly.Graph titration curve as absorbance(ordinate) vs. volume of biotin added(abscissa). Note the volume (l) of biotinadded at the inflection point of the curve.The amount of biotin at the inflection pointis divided by the concentration of thestreptavidin sample to yield the specificactivity.Calculation1 Streptavidin + 4 biotin Streptavidin(biotin) 4The formation of the complex is measured at233 nm.ReagentsAmmonium Carbonate Buffer:200 mM (NH 4) 2CO 3, pH 8.91 mg/ml solution of d-biotin (dissolved inAmmonium Carbonate Buffer)10 mg/ml Streptavidin sample (dissolved inde-ionized water)ProcedureAdjust spectrophotometer to read at 233 nm.Dilute the dissolved streptavidin sample to0.1 mg/ml in Ammonium CarbonateBuffer. Prepare at least 1 ml.where:V 1 = vol of d-biotin at inflection pointC 1 = concentration of d-biotin solutionV 2 = volume of streptavidin sampleC = concentration of streptavidin sample2SUGGESTIONS FOR USEMeasurementBecause the lyophilizate is ~10% NaCl, wesuggest that the customer dissolve it andmeasure it using its extinction coefficient{E (1%) = 32} rather than weighing it.280

Aliquots may be stored at -20C for longperiods. If kept refrigerated, an antimicrobialagent such as 0.05% sodium azide should beadded to retard growth and subsequentproteolysis from exogenous enzymes.SolubilityBayer (1989) reports that streptavidin mayform aggregates under certain conditions.There is a tendency for lyophilizedstreptavidin to aggregate when it isredissolved in water or other low ionicstrength buffers at neutral or acidic pH.REFERENCESBayer, E. A., Ben-Hur, H., Hiller, Y. and M. Wilchek.Postsecretory Modifications of Streptavidin. Biochem J259: 369–376 (1989).Green, N. M. Spectrophotometric Determination of Avidinand Biotin, in Meth Enzymol 18 (McCormick, D. B.and L. D. Wright, eds.) Academic Press, New York,p. 418 (1970).ProZyme Streptavidin-plus has beenlyophilized from a dilute sodium chloridesolution at mildly alkaline pH in order tominimize aggregate formation. This materialis readily soluble in water or salt-containingbuffers, up to 50 mg/ml or more.However, in rare cases Streptavidin-plus maycontain a small amount of insoluble materialwhen dissolved in de-ionized water or lowionic strength buffers, either at the time it isdissolved or after freezing and re-thawing.This effect is generally not seen in thepresence of salt-containing buffers such asPBS.If undissolved material is observed, it can beremoved by centrifugation and does notconstitute a significant fraction of the totalprotein. The activity of the materialrecovered after reconstitution under theseconditions is undiminished.

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