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Tissue samples- After cutting samples, check the weight,add PBSPH7.2-7.4 , Rapidly frozen with liquid nitrogen, maintain samples at 2-8! after melting,add PBSPH7.4 , Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant. Assay procedure 1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100l to the first and the second well, then add Standard dilution 50l to the first and the second well, mix; take out 100l form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50l to the third and the forth well ,mix ; then take out 50l from the third and the forth well discard, add 50l to the fifth and the sixth well ,then add Standard dilution 50l to the fifth and the sixth well, mix ; take out 50l from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50l to the seventh and the eighth well ,mix ; take out 50l from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50l to the ninth and the tenth well, mix , take out 50l from the ninth and the tenth well discard(add Sample 50l to each well after Diluting ,(density: 300 pg/mL,200pg/mL ,100pg/mL,50pg/mL,25pg/mL) 2.add sampleSet blank wells separately (blank comparison wells don t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40l to testing sample well, then add testing sample 10l (sample final dilution is 5-fold), add sample to wells , don t touch the well wall as far as possible, and Gently mix. 3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37!. 4.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve. 5.washingUncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat. 6.add enzymeAdd HRP-Conjugate reagent 50l to each well, except blank well. 7.incubateOperation with 3. 8.washingOperation with 5. 9.colorAdd Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37! 10.Stop the reactionAdd Stop Solution50l to each well, Stop the reaction(the blue color change to yellow color). 11.assaytake blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min. Important notes The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley . if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.n5 . Closure plate membrane only limits the disposable use, to avoid cross-contamination. The substrate evade the light preservation. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard. All samples, washing buffer and each kind of reject should according to infective material process. Do not mix reagents with those from other lots. Calculate  Assay range 15pg/mL  400pg/mL Storage and validity 1Storage 2-8!. 2validity six months.     PAGE  PAGE 1  Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.  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