Human P53 protein FOR RESEARCH USE ONLY Drug Names Generic Name:Human P53 protein (P53) ELISAKit. Purpose This kit allows for the determination of P53 concentrations in Human serum, blood plasma, and other biological fluids. Principle of the assay T he kit assay Human P53 level in the sample,use Purified Human P53 antibody to coat microtiter plate wells, make solid-phase antibody, then add P53 to wells, Combined P53 antibody which With HRP labeled , become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of P53 in thesamples is then determined by comparing the O.D. of the samples to the standard curve. Materials provided with the kit Materials provided with the kit 48determinations 96 determinations Storage User manual 1 1 Closure plate membrane Sealed bags 1 1 Microelisa stripplate 1 1 2-8℃ Standard :45μg/mL 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃ Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8℃ HRP-Conjugate reagent 3ml×1 bottle 6ml×1 bottle 2-8℃ Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8℃ Chromogen SolutionA 3ml×1 bottle 6ml×1 bottle 2-8℃ Chromogen SolutionB 3ml×1 bottle 6ml×1 bottle 2-8℃ Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃ wash solution (20ml×20 fold ) ×1 bottle (20ml×30 fold ) ×1 bottle 2-8℃ Specimen requirements 1. serum- coagulation at room temperature 10-20 mins,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. 2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugalagain. 3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it. 4. cell culturesupernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBS(PH7.2-7.4), Cell concentration reached 1 million /ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min atthe speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. 5. Tissue samples-After cutting samples, check theweight,add PBS(PH7.2-7.4), Rapidly frozen with liquid nitrogen, maintain samples at 2-8℃ after melting,add PBS(PH7.4), Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant. 6. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction.If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeatedfreeze-thaw cycles. 7. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active. Assay procedure 1.Dilute and add sample to Standard:set 10 Standard wells on the ELISA plates coated, add Standard 100μl to the first and the second well, then add Standard dilution 50μl to the first and the second well,mix; take out 100μl form the first and the second well then add it to the third and the forthwell separay. then add Standard dilution 50μl to the third and the forth well ,mix ; then take out 50μl from the third and the forth welldiscard, add 50μl tothe fifth and the sixth well ,then add Standarddilution 50μl to the fifth and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add to the seventh and the eighth well, then addStandard dilution 50μl to the seventh and the eighth well ,mix ; takeout 50μl from the seventh and the eighth well and add to the ninthand the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out50μl from the ninth and the tenth well discard(add Sample 50μl to eachwell after Diluting ,(density: 30μg/mL,20μg/mL,10μg/mL, 5μg/mL, 2.5μg/mL) 2.add sample:Set blank wells separay (blank comparison wells don’t add sample and HRP-Conjugate reagent,other each step operation is same). testing sample well. add Sample dilution40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sampleto wells , don’t touch the well wall as far as possible, and Gently mix. 3.add enzyme:Add HRP-Conjugate reagent 50μl toeach well, except blank well. 4.Incubate:After closing plate with Closure plate membrane ,incubate for 30 min at 37℃. 5.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve. 6.washing:Uncover Closure plate membrane,discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5times, dry by pat. 7.color:Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 10 min at 37℃ 8.Stop the reaction:Add Stop Solution50μl toeach well, Stop the reaction(the blue color change to yellow color). 9.assay:take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min. Important notes 1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not useup after opened,the plate should be stored in Sealed bag. 2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result. 3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 mins, if the number of sample is much , recommend to use Volley . 4. if the testing materialcontent is excessively higher (The sample OD is bigger than thefirst standard well ),please dilute Sample (n-fold), Please diluenteand multiplied by the dilution factor.(×n×5). 5. Closure plate membrane only limits the disposable use, to avoid cross-contamination. 6. The substrate evade the light preservation. 7. Please according to use instruction strictly, The testresult determination must take the microtiter plate reader as a standard. 8. All samples, washing buffer and each kind of reject should according to infective material process. 9. Do not mix reagents withthose from other lots. Calculate Assay range 1.6μg/mL -40μg/mL Storage and validity 1.Storage: 2-8℃. 2.validity: six months. Takethe standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the correspondingdensity according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equationof the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate thesample density, multiplied by the dilution factor, the result is the sample actual density. This chart for reference only