3.Methods 3.1 Prepare the standard curve Take 7 tubes and do as following table: Tube No. Reagents 1 2 3 4 5 6 7 Standard(ml) 010 020 040060 0.801.00 H2O(ml) 1.000.90 0.80 0.60 0.40 0.20 Complex-forming reagent 5.00 5.00 5.00 5.00 5.00 5.00 5.00 (ml) Mix up and stand at room temperature(RT)for 10 min Folin reagent(ml) 0.500.500.500.500.500.500.50 Mix up the solution immediately,react at 30'C(or RT)for 30 min,Set No.1 tube as control(0) measure OD at 500nm 3.2 Determine the sample's concentration Transfer 0.20 ml sample protein solution into a clean tube,add 0.80 ml distilled H2O,then measure the mixed solution's ODsoo according to the above protocol. 4.Results 4.1 Set the standard protein's concentration as X-axis.OD500 as Y-axis.draw a standard curve. 4.2 Read the sample's concentration in the standard curve savailable as a precipitate,then dissolve the precipitate in 2 Hydrolyze at 100'C for 10 min in a heating block or boiling water bath.Carry 0.2-mL aliquots of the hydrolyzate.cool to room temperature and add I mL of freshly mixed complex-forming reagent.Let the solution stand at room tem perature for 10min. 2.The reaction is v pH dependent,and it therefor mportant to maintain the pH between10 and 10.5.Therefore.take are when analyzing sa that are in stronbuffer outside this range 3.The vortex-mixing step is critical for obtaining reproducible results.The Folin reagent is reactive only for a short time under these alkaline conditions,being unstable in alkali,and great care should therefore be taken thoroug mixing. 4.The assay is not line at higher ion.Ensure that you are your sampon the linear portion of the calibration curve. 5.A set of standards is needed with each group of assays,preferably in duplicate.Duplicate or triplicate unknowns are recommended. 6.One disadvantage of the Lowry method is the fact that a range of substanees interferes with this includin s.drugs, ucleic acids.d sugars.(Th effect of some of these agents is shown in Table I in Chapter 3.)In many cases.the effects of these agents can be minimized by diluting them out,assuming that the protein concentration is sufficiently high to still be detected after dilution.When interfering compounds are involved,it is,of course.important to run an