Precision Plus Protein WesternC standards.pdf

Precision Plus Protein WesternC standards areintended for use in a wide range of applications andallow easy visualization during electrophoresis,blottransfer,and chemiluminescent development of westernblots on film or by CCD imagers.ApplicationUseGel electrophoresisMonitoring migration duringelectrophoresisBlot transferAssessing transfer efficiencyBlot developmentEstimating molecularweight on film and CCDimagersRelated Products for Blot Detection Catalog#Description161-0385Precision Plus Protein WesternC Pack,(Precision Plus Protein WesternC standards,StrepTactin-HRP Conjugate,0.125 ml)161-0380Precision Protein StrepTactin-HRP Conjugate,0.3 ml161-0381Precision Protein StrepTactin-HRP Conjugate,0.125 ml170-5070Immun-Star WesternCChemiluminescent Kit170-5044Immun-Star GAM-HRP Detection Kit170-5045Immun-Star GAR-HRP Detection Kit170-5047Immun-Star GAM-HRP Conjugate,2 ml170-5046Immun-Star GAR-HRP Conjugate,2 ml170-5010Immun-Star AP GAM-AP Detection Kit170-5011Immun-Star AP GAR-AP Detection Kit161-0382Precision Plus StrepTactin-AP Conjugate,0.3 mlInstruction ManualPrecision PlusProteinWesternCStandardsCatalog#161-0376Product shipped at 4C.Store at 20C upon arrival.Bio-Rad Laboratories,Inc.2000 Alfred Nobel Dr.,Hercules,CA 94547 USA(510)741-100010008761 Rev BA representative lot ofPrecision Plus ProteinWesternC standards ona Criterion 420%Tris-HCl gel and awestern blot detectedby chemiluminescence25371015205075100150250kDGelBlot10008761B.qxp 3/4/2008 10:29 AM Page 1Precision Plus Protein WesternC standards areintended for use in a wide range of applications andallow easy visualization during electrophoresis,blottransfer,and chemiluminescent development of westernblots on film or by CCD imagers.ApplicationUseGel electrophoresisMonitoring migration duringelectrophoresisBlot transferAssessing transfer efficiencyBlot developmentEstimating molecularweight on film and CCDimagersRelated Products for Blot Detection Catalog#Description161-0385Precision Plus Protein WesternC Pack,(Precision Plus Protein WesternC standards,StrepTactin-HRP Conjugate,0.125 ml)161-0380Precision Protein StrepTactin-HRP Conjugate,0.3 ml161-0381Precision Protein StrepTactin-HRP Conjugate,0.125 ml170-5070Immun-Star WesternCChemiluminescent Kit170-5044Immun-Star GAM-HRP Detection Kit170-5045Immun-Star GAR-HRP Detection Kit170-5047Immun-Star GAM-HRP Conjugate,2 ml170-5046Immun-Star GAR-HRP Conjugate,2 ml170-5010Immun-Star AP GAM-AP Detection Kit170-5011Immun-Star AP GAR-AP Detection Kit161-0382Precision Plus StrepTactin-AP Conjugate,0.3 mlA representative lot ofPrecision Plus ProteinWesternC standards ona Criterion 420%Tris-HCl gel and awestern blot detectedby chemiluminescence25371015205075100150250kDGelBlot10008761B.qxp 3/4/2008 10:29 AM Page 1Product DescriptionPrecision Plus Protein WesternC standards offer 10distinct,prestained protein bands with a molecularweight of 10,15,20,25,37,50,75,100,150,and250 kD.The 25,50,and 75 kD bands are stainedpink for easy band referencing and blot orientation.Precision Plus Protein WesternC standards contain anintegral Strep-tag sequence,enabling parallel detectionon western blots using StrepTactin-HRP(catalog#161-0380,161-0381)or StrepTactin-AP(catalog#161-0382)and HRP or AP-conjugated secondary antibodies specific to the protein of interest.The molecular weights of the Precision Plus ProteinWesternC standards are confirmed by migration in aLaemmli SDS-PAGE system.The sharp ladder bandsprovide an accurate estimation of the molecularweight of unknown proteins,with an R2value 0.99on a 420%Tris-HCl SDS-PAGE gel.*Instructions for UsePrecision Plus Protein WesternC standards are provided in loading buffer and require no dilution.Before use,allow the tube to reach room temperature and mix thoroughly.This will ensure thatany solids that may have precipitated at 20C havereturned to solution.Do not heat the product aboveroom temperature.For chemiluminescent detection of the PrecisionPlus Protein WesternC standards:1.Load 5 l of the Precision Plus ProteinWesternC standard onto the gel next to protein of interest2.Run the SDS-PAGE gel at the appropriate voltage,current and time3.Transfer the protein to a membrane at theappropriate voltage and current4.Block the membrane in blocking solution5.Probe with a primary antibody specific to the protein of interest 6.Wash the blot*Migration pattern of the 25,50,and 75 kD bands may differ onneutral pH gels(Criterion XT gels or similar).7.Probe with a secondary antibody appropriateto the protein of interest,and include approximately 1 l of StrepTactin-HRP1orStrepTactin-AP per 10 ml of secondaryincubation solution to detect the bands of thePrecision Plus Protein WesternC standards8.Wash the blot 9.Develop with a suitable development reagent,such as the Bio-Rad Immun-Star HRP kit orImmun-Star AP kit(for film detection),orImmun-Star WesternC kit(for CCD imaging)Note:The recommended dilution of theStrepTactin-HRP and StrepTactin-AP conjugateis 1:10,0001:50,000 for chemiluminescent detection2with the Immun-Star WesternC kit(#170-5070 or similar).SpecificationsCatalog#161-0376Volume250 lNumber of 50applicationsLoading buffer30%(w/v)glycerol,2%SDS,62.5 mM Tris,pH 6.8,50 mM DTT,5 mM EDTA,0.02%NaN3Shelf lifeMinimum 12 months at 20C;if theproduct has been stored as directedfor longer than 12 months,the shelflife may be extended by adding dithiothreitol(DTT)to approximately 50 mMRecommended Loading VolumesLoad Volume3Application5 lChemiluminescent blot developmentStrep-tag technology for western blot detection is covered byUS patent 5,506,121 and by UK patent 2,272,698.StrepTactinis covered by German patent application P 19641876.3.Bio-RadLaboratories,Inc.is licensed by Institut fr Bioanalytik GmbH tosell these products for research use only.1StrepTactin-HRP and StrepTactin-AP are designed not to interfere with secondary antibody incubation.2For longer exposure times,use of a greater dilution ofStrepTactin-HRP(1:50,0001:100,000)will prevent overexposure of the bands of the Precision Plus ProteinWesternC standards.3 For very short exposure times,or for enhanced band intensityon a gel or a blot,an optional load volume of 10 l may beused.10008761B.qxp 3/4/2008 10:29 AM Page 3