豬磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶（pERK）試劑盒說(shuō)明書(shū)

本試劑盒用于測(cè)定豬血清，血漿及相關(guān)液體樣本中磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶 （Perk）的活性。

（pERK）實(shí)驗(yàn)原理：

本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中豬磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶 （pERK）水平。用純化的豬磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶（pERK）抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶（pERK），再與HRP標(biāo)記的磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶（pERK）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過(guò)*洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成zui終的黃色。顏色的深淺和樣品中的磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶（pERK）呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中豬磷酸化細(xì)胞外信號(hào)調(diào)節(jié)激酶（pERK）濃度。

試劑盒組成：

樣本處理及要求：

1. 血清：室溫血液自然凝固10-20分鐘，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過(guò)程中如出現(xiàn)沉淀，應(yīng)再次離心。

2. 血漿：應(yīng)根據(jù)標(biāo)本的要求選擇EDTA或檸檬酸鈉作為抗凝劑，混合10-20分鐘后，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過(guò)程中如有沉淀形成，應(yīng)該再次離心。

3. 尿液：用無(wú)菌管收集，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過(guò)程中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照實(shí)行。

4. 細(xì)胞培養(yǎng)上清：檢測(cè)分泌性的成份時(shí)，用無(wú)菌管收集。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。檢測(cè)細(xì)胞內(nèi)的成份時(shí)，用PBS（PH7.2-7.4）稀釋細(xì)胞懸液，細(xì)胞濃度達(dá)到100萬(wàn)/ml左右。通過(guò)反復(fù)凍融，以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。保存過(guò)程中如有沉淀形成，應(yīng)再次離心。

5. 組織標(biāo)本：切割標(biāo)本后，稱(chēng)取重量。加入一定量的PBS，PH7.4。用液氮迅速冷凍保存?zhèn)溆谩?biāo)本融化后仍然保持2-8℃的溫度。加入一定量的PBS（PH7.4），用手工或勻漿器將標(biāo)本勻漿充分。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。分裝后一份待檢測(cè)，其余冷凍備用。

6. 標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行，提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于-20℃保存，但應(yīng)避免反復(fù)凍融.

7. 不能檢測(cè)含NaN3的樣品，因NaN3抑制辣根過(guò)氧化物酶的（HRP）活性。

試劑盒性能：

1.樣品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值為0.92以上。

2.批內(nèi)與批間應(yīng)分別小于9%和15%

保存條件及有效期：

1.試劑盒保存：2-8℃。

2．有效期：6個(gè)月

FOR RESEARCH USE ONLY

Drug Names

Generic Name：Porcine pERK ELISA Kit.

Purpose

This kit allows for the determination of pERK in Porcine serum, plasma, and other biological fluids.

Principle of the assay

The kit assay Porcine pERK level in the sample，use Purified Porcine pERK antibody to coat microtiter plate wells, make solid-phase antibody, then add pERK o wells, Combined pERK 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 pERK in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Materials provided with the kit

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 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.

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 culture supernatant-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 at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.

5.       Tissue samples- After cutting samples, check the weight,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 repeated freeze-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 forth well separay. then add Standard dilution 50μl to the third and the forth well ,mix ; then take out 50μl from the third and the forth well discard, add 50μl to the fifth and the sixth well ,then add Standard dilution 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 add Standard dilution 50μl to the seventh and the eighth well ,mix ; take out 50μl from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth well discard（add Sample 50μl to each well after Diluting ,（density: 90 U/L，60 U/L，30 U/L，15 U/L，7.5 U/L）

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 dilution 50μl to testing sample well, then add testing sample 10μl （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.washing：Uncover 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 enzyme：Add HRP-Conjugate reagent 50μl to each well, except  blank well.

7.incubate：Operation with 3.

8.washing：Operation with 5.

9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃

10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color change to yellow color）.

11.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 use up 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 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.（×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 test result 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 with those from other lots.

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.