BCA蛋白定量Protein Assay KitIntroduction and product overview:Protein quantification is often required before precessing protein samples for analysis.The Bicinchoninic Acid (BCA) Protein Assay is highly sensitive colorimetric assay that isnot affected by chemicals in the sample. The BCA Protein Assay primarily reduces Cu2+to Cu1+ by proteins in an alkaline environment followed by highly sensitive and selectivecolorimetric detection of BCA/copper complex. It is water-soluble and strongly absorbslight at 562nm in a linear fashion with increasing protein concentration.Kit contents:Biogot BCA Reagent A: 100ml, stored at room temperatureBiogot BCA Reagent B: 2ml stored at room temperatureBovine Serum Albumin Standard: 100mg(Power), Stored at 4℃.( Recommend to makestock solution(2mg/ml) and aliquot before store at 4℃ )Test Tube Procedures:A) Standard Preparation:Label 9 test tubes with A-I and prepare the standards as indicated below. The diluentused should be the same as used for the protein samples. The following dilutions aresuitable for duplicate Standard assays.Tube Bovine Serum Albumin Diluent(μl) Final Concentration(μg/ml)A 200μl from Stock 0 2,000B 120μl from Stock 40 1,500C 100μl from Tube A 100 1,000D 100μl from Tube B 100 750E 100μl from Tube C 100 500F 100μl from Tube E 100 250G 100μl from Tube F 100 125H 20 μl from Tube G 80 25I 0 100 0(Blank)B) Working Solution Preparation:1) Use following formula to determine the amount of working solution required.(Total number of standards and samples)*(Number of replicates)*(Volume ofworking solution sample)= Total volume working solution required.2) Mix fifty parts of BCA Reagent A with one part of BCA reagent B (50:1, ReagentA:B).C) Micro-plate Procedure (Sample to WR ratio = 1:20)1. Pipette10μlofeachstandardorunknownsamplereplicateintoamicro-platewell(workingrange=20-2,000μg/ml).2.Add200μloftheWRtoeachwellandmixplatethoroughlyonaplateshakerfor30seconds.3.Coverplateandincubateinthewaterbathat37°Cfor30minutes.4.CoolplatetoRT.5.Measuretheabsorbencyatornear562nmonaplatereader.6.Usethestandardcurvetocalculatetheproteinconcentrationofeachunknownsample.Notes1)Certainsubstancesincludingreducingpotential,chelatingagents,andstrongacidsorbasesareknowntointerferewithproteinestimationandavoidthosesubstancesinthesamplebuffer.(Forexample,EDTA,EGTA,DTT)2)PrepareaclearandfreshWRreagentatroomtemperaturewhenpreppingnewexperiments.AfteraddingWRreagent,itcouldbeincubatedforsixtyminutesat37°Cor2hoursatroomtemperature.Absorbanceat562nmincreaseswiththeincreasingincubationtime.Colordevelopmentrunsfasterwiththeincreasingtemperature.Ifsampleconcentrationistoolow,itwillbebettertorunthereactionatahighertemperatureorincreasetheincubationtime.3)Goodlinearrangeforsamplesisfrom50-2000μg/ml.4)BCAassayisinterferedwithbychelatingagentsandhighconcentrationreducingagents.MakesureEDTA<10mM,noEGTA,DTT<1mMandβ-ME<1mMininthesamplebuffer.Trytoremovetheinterferingsubstancebydialysisorgelfiltrationtoeliminateorminimizetheeffectsofinterferingsubstances.Ifinterferencecannotbeovercome,itisrecommendyouusetheBradfordproteinassaykit.5)periodofvalidityis6months.