PCNA核内参抗体说明书
核蛋白内参
核蛋白内参我们知道，内参蛋白是指由管家基因（house keeping gene）编码表达的一类蛋白，它们在各组织和细胞中的表达相对恒定，在实验中检测蛋白的表达水平变化时常用它们来做参照物。
这些内参蛋白常应用于 Western Blot 中，Western Blot 不仅能证明样品中是否含有某种蛋白，还能比较不同条件、不同组织和细胞中目的蛋白的相对含量，从而衡量蛋白的表达水平。
内参蛋白作为一种参照物，在其中的作用为确定样本使用量的一致性，以及校正实验过程中可能出现的操作误差，从而进一步保证实验结果的准确性。
那么对于不同的蛋白，内参蛋白该如何选择呢？前两节介绍了几种常见内参蛋白的选择和应用。
本节将系统介绍不同内参蛋白的选择以及云克隆公司现有的内参蛋白抗体。
对于内参蛋白的选择首先是考虑分子量。
一般情况下，内参蛋白和检测的目的蛋白最好相差在 5kDa 以上。
如分子量为45kDa 的检测蛋白，我们可以选择 Tubulin Beta（54kDa），Lamin B1（66kDa）等，而不选择 ACTa2（42kDa）或者 beta-actin（45kDa）。
其次对于不同的组织，应选择不同的内参。
如肌动蛋白是一种细胞骨架蛋白，有六种不同的亚型，包括： alpha-skeletal muscle actin、alpha-cardiac muscle actin、alpha-smooth muscle actin、gamma-smooth muscle actin、beta-actin (β-non-muscle) 和 gamma-non-muscle actin。
其中常用作Western Blot 内参的 beta-actin 在肌肉和脂肪组织中分布较少，那么若样本是心肌或脂肪组织来源的，就可以考虑分别用 alpha-cardiac muscle actin (ACTC1) 和 GAPDH 作为内参蛋白。
最后，提取蛋白的部位不同，选择的内参蛋白也不同。
Roche MagNA Lyser 产品说明书
Ordering Information Cat. No. Product ***********MagNA Lyser Instrument (230 Volt)***********MagNA Lyser Instrument (110 Volt)(Instruments supplied with rotor and rotor cooling block)***********MagNA Lyser Green Beads (100 tubes)Related Products Cat. No. Product***********MagNA Pure LC DNA Isolation Kit II (Tissue)***********MagNA Pure LC mRNA Isolation Kit II (Tissue)03 330 591 001MagNA Pure LC RNA Isolation Kit III (Tissue)***********MagNA Pure LC DNA Isolation Kit III (Bacteria, Fungi)***********MagNA Pure LC RNA Isolation Tissue Lysis Buffer – Refill (70 ml)System DescriptionHomogenize up to 16 samples in just a few seconds.Save valuable lab space with a small benchtop instrument.Reduce hands-on time by replacing the mortar and pestle and other manual methods.Integrate your workflow with the automated nucleic acid isolation of the MagNA Pure LC Instrument.Perform consistent and reproducible sample disruption.Process many different sample types.Prevent nucleic acid degradation with the benchtop cooling unit.Ease your setup with a removable rotor and prefilled disposable vials.Automate with an easy-to-use instrumentVersatile, efficient, and rapid pre-preparationFigure 71. Add your sample and lysis buffer to the MagNA Lyser Green Beads.2. Homogenize with the MagNA Lyser Instrument.3. Centrifuge to pellet the debris.4. Proceeed with the supernatant to prepare nucleic acids or proteins.For detailed information,visit or contact your local representative.Trademarks:MagNA Pure, MagNA Lyser, LightCycler, and the MagNA Pure Logo are trademarks of a member of the Roche Group.The technology used for the LightCycler System is licensed from Idaho Technology Inc., Salt Lake City, UT, USA.Fully automated sample preparationon the PCR Workflow SystemRoche Diagnostics GmbH Roche Applied Science Nonnenwald 282372 Penzberg Germany0000Roche Applied Science Part of Roche DiagnosticsMagNA Lyser InstrumentStart the Ball Rollingwith Automated Tissue HomogenizationᕤᕣᕢᕡFigure 6Components of the system.The MagNA Lyser InstrumentAutomated tissue homogenizationProcessing conditionsRefer to the following tables for guidelines on setting up your homogenizationSample material(10 mg)*Time settings(seconds)Cooling(between the runs)Speed Average yield(µg)***Average purity(OD 280/260 nm)***Spleen 2 x 25 906,00030–40 1.9Liver 25-6,00016–18 1.8Lung 2 x 25906,00025 1.8Kidney25-6,000201.8Maize leaves **20-5,00010n.d.Maize polenta **20-5,0008n.d.Tortilla chips **20-5,0001n.d.*Aliqout containing 10 mg sample material (here mouse and food samples) was taken for the DNA purificationusing the MagNA Pure LC DNA Isolation Kit II (Tissue), (see pack insert)**Centrifugation after the homogenization for 5 minutes at 2,200 x g*** Yield and purity strongly depend on the condition of the sample material n.d.not determinedData kindly provided by Dr. Peterhänsel, RWTH Aachen, GermanyFigure 1Gel electrophoresis from genomic DNA isolated from tissue homogenized with the MagNA LyserInstrument, using the MagNA Pure LC DNA Kit II (Tissue).Marker: DNA Marker III*Aliquot containing 10 mg sample material (here mouse and human research samples) was taken to purify RNAeither with the MagNA Pure LC RNA Isolation Kit III (Tissue) or the MagNA Pure LC mRNA Isolation Kit II (Tissue) homogenized with the MagNA Lyser Instrument.** Yield and purity strongly depend on the condition of the sample material. The yield for mRNA was not determined.Sample material(10 mg)*Time settings(cycles/seconds)Cooling(between/afterthe runs in seconds)SpeedAverage yield (mg)(total RNA)**Average purity(OD 280/260 nm)**RNA/mRNARarely expressed targets in small numbers of target cells,as seen in experiments about minimalresidual diseases,are difficult to detect.Increasing the cell number can improve sensitivity and lead to accurate results.Without the MagNA Lyser pre-processing,the MagNA Pure mRNA HS Kit can efficiently obtain mRNA from a maximum of 1 x 107white blood cells (WBCs),as shown in research studies with human samples.However,using greater cell numbers results in a saturation effect with quantitative assays (Figure 3).Homogenization of the lysate with the MagNA Lyser Instrument prior to the purification eliminatesthe amplification saturation at 1 x 107cells and allows the use of up to 2.5 x 107WBCs (Figure 4 and 5),enhancing the analytical sensitivity of the assay.Eliminate sensitivity barriers with increased sample inputFigure 3mRNA was purified from different amounts of human white blood cells with the MagNA Pure mRNA HS Kit. G6PDH was amplified using the LightCycler t(9;22) Quantification Kit (see text beside).Figure 4mRNA was purified from different amounts of human white blood cells with the MagNA Pure mRNA HS Kit. The lysates from 2.5 x 107cells and 5 x 107cells were homogenized with the MagNA Lyser Instrument (2x50 seconds with 90 seconds cooling in between) prior to the mRNA purification. G6PDH was amplified using the LightCycler t(9;22) Quantification Kit (see text beside).Figure 5Scalability from 1 x 106cells to 2.5 x 107cells is represented in the graph and the table of the relationship between crossing points and cell numbers. The limitation of cell input is indicated by no change in crossing point with increased cell number (see text beside).Cell number 5 x 1072.5 x 1071 x 1075 x 1061 x 106Log (cell number)7.77.47.06.76.0Crossing point 20.320.321.822.424.4crossingpointLog(cell number)252423222120195.86.36.87.37.8Figure 2Gel electrophoresis from total RNA isolated from tissue homogenized with the MagNA Lyser Instrument, using the MagNA Pure LC RNA Kit III (Tissue).Ma r k e rS p l e e nL i v e rL u n gK i d n e yM a r k e rMa i z e l e a v e sMa i z e l e a v e sS p l e e nL i v e r11 kb5 kb5 kb28 S rRNA 18 S rRNASpleen 2 x 50 90 6,500–7,000 30–40 1.9Liver 50 - 6,500–7,000 13–17 2.0Thymoid tissue60906,500n.d.n.d.Heart 60 90 6,500 n.d. n.d.Abdominal fat 60 90 6,500 n.d. n.d.Aorta 60 90 6,500 n.d. n.d. Other samples1+n x 50 90 6,500–7,000- -1 x 105 x 101 x 10- 5 x 101 x 105 x 105 x 10- 5 x 102.5 x 10 5 x 10。
本试剂仅供研究使用
本试剂仅供研究使用人抗增殖细胞核抗原抗体(PCNA)elisa试剂盒使用说明书试剂盒组成：48孔配置/96孔配置说明书：1份封板膜：2片（48）/2片（96）密封袋：1个目的：【人抗增殖细胞核抗原抗体(PCNA)elisa试剂盒说明书】本试剂盒用于测定人血清，血浆及相关液体样本中介素2(IL-2)的含量。
服务承诺：供货期：款到发货。
工作时间内免费的技术咨询和指导。
请来电咨询为客户提供来样检测服务，最大限度实验结果的有效性(免费代测)。
试剂盒性能：1.样品线性回归与预期浓度相关系数R值为0.95以上。
2.批内与批见应分别小于9%和11%保存条件及有效期：1.试剂盒保存：2-8℃。
2．有效期：6个月检测范围：0.2IU/L - 6IU/L实验原理：【人抗增殖细胞核抗原抗体(PCNA)elisa试剂盒说明书】本试剂盒应用双抗体夹心法测定标本中人抗增殖细胞核抗原抗体(PCNA)水平。
用纯化的人抗增殖细胞核抗原抗体(PCNA)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入介素2(IL-2)，再与HRP标记的介素2(IL-2)抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。
TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。
颜色的深浅和样品中的介素2(IL-2)呈正相关。
用酶标仪在450nm波长下测定吸光度（OD 值），通过标准曲线计算样品中人抗增殖细胞核抗原抗体(PCNA)浓度。
样本处理及要求：1. 血清：室温血液自然凝固10-20分钟，离心20分钟左右（2000-3000转/分）。
仔细收集上清，保存过程中如出现沉淀，应再次离心。
2. 血浆：应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂，混合10-20分钟后，离心20分钟左右（2000-3000转/分）。
仔细收集上清，保存过程中如有沉淀形成，应该再次离心。
3. 尿液：用无菌管收集，离心20分钟左右（2000-3000转/分）。
壮骨健膝方对脂多糖诱导兔滑膜成纤维细胞炎症模型的影响
·实验研究 ·福建中医药 2023 年 6 月 第 54 卷 第 6 期Fujian Journal of TCM June 2023，54（6）壮骨健膝方对脂多糖诱导兔滑膜成纤维细胞炎症模型的影响张英杰1，张鹏1，肖艳2，刘俊1，陈雨1，邱梦婷1，苏友新1*（1.福建中医药大学中医学院，福建 福州 350122；2.福建中医药大学康复医学院，福建 福州 350122）摘要： 目的 探讨壮骨健膝方对兔滑膜成纤维细胞炎症模型的影响及作用机制。
方法 采用脂多糖（LPS ）刺激兔滑膜成纤维细胞（FLS ）建立炎症模型，筛选壮骨健膝方干预炎症模型的最佳条件（浓度10%，时间48h ）后，采用随机数字表法随机分为空白组、模型组和壮骨健膝方组，分别给予相应条件干预后采用ELISA 法检测各组细胞上清液中白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）含量，qPCR 法检测各组细胞中IL-1β、TNF-α、IL-6、增殖细胞核抗原（PCNA ）、I κB α、NF-κB p65 mRNA 表达，Western blot 法检测各组细胞中PCNA 、核因子κB 抑制因子α（I κB α）及核内核因子κB （NF-κB ）p65蛋白表达。
结果 与空白组比较，模型组IL-1β、TNF-α、IL-6含量，IL-1β、TNF-α、IL-6、PCNA 、NF-κB p65 mRNA 及核内NF-κB p65蛋白表达均显著升高（P 均＜0.05），I κB α mRNA 及蛋白表达显著降低（P 均＜0.05）；与模型组比较，壮骨健膝方组IL-1β、TNF-α、IL-6含量，IL-1β、TNF-α、IL-6、PCNA 、NF-κB p65 mRNA 及核内NF-κB p65蛋白表达均显著降低（P 均＜0.05），I κB α mRNA 及蛋白表达显著升高（P 均＜0.05）。
免疫试验必备 内参抗体
品名 货号 价格 宿主
应用
His, HRP Labeled
CB101001 ￥3000/100ug
Rabbit
WB 1:1000 IHC 1:200 IP 1:200
北京西美杰科技有限公司 上海 电话：021-63599871 传真：021-63599873 Email：shanghai@
货号 25-01-01 24-01-01 25-00-01 24-00-02 24-00-01
品名 HisDetector Nickel-AP HisDetector Nickel-HRP HisDetector Western Blot Kit, AP Colorimetric HisDetector Western Blot Kit, HRP Chemiluminescent HisDetector Western Blot Kit, HRP Colorimetric
检测物种 Human, Mouse, Rat
品名 货号 价格 宿主
Rubisco Large Subunit AS03 037 ￥4770/100ug Rabbit
应用
WB 1:5000-1:10000 IF 1: 250
检测物种 plant and algal
北京西美杰科技有限公司 上海 电话：021-63599871 传真：021-63599873 Email：shanghai@
品名 货号 价格 宿主
β-actin CB100996 ￥1500/100ug Rabbit
应用
WB 1:1000 IP 5ug/mg lysate
检测物种 Human, Mouse
品名 货号 价格
β-actin 60008-1-Ig ￥1548/150ul
All-in-One qPCR Mix 产品说明书
All-in-One™qPCR MixFor universal quantitative real-time PCRCat.No.QP001(Old Cat.No.AOPR-0200,20μl×200reactions)Cat.No.QP002(Old Cat.No.AOPR-0600,20μl×600reactions)Cat.No.QP004(Old Cat.No.AOPR-1000,20μl×1000reactions)Cat.No.QP005(Old Cat.No.AOPR-4000,20μl×4000reactions)Performance optimized for All-In-One™qPCR Primers,All-In-One™miRNA qPCR Primers, miProfile™miRNA qPCR Arrays,ExProfile™Gene qPCR Arrays,All-In-One™First-Strand cDNA Synthesis Kit and All-In-One™miRNA First-Strand cDNA Synthesis KitUser ManualGeneCopoeia,Inc.9620Medical Center Drive,#101Rockville,MD20850USA301-762-0888866-360-9531***********************©2016GeneCopoeia,Inc.USER MANUALAll-in-One TM qPCR MixI.DescriptionII.Related ProductsIII.Contents and StorageIV.PreparationV.ProcedureVI.ExampleVII.Trouble Shooting GuideVIII.Limited Use License and WarrantyI.DescriptionThe All-in-One™qPCR Mix provides fast and efficient SYBR®Green-based real-time quantitative PCR.The qPCR Mix uses a high-fidelity hot-start DNA polymerase,optimized reaction buffer and high-quality dNTPs to enable specific and sensitive amplification of even low-copy genes or miRNAs.The All-in-One TM qPCR Mix reduces experimental design time by providing a universal reaction condition that can be used with almost all primers and most real-time PCR instruments.II.Related ProductsGeneCopoeia offers comprehensive solutions for studying gene expression.A careful process of co-development ensures that they work well together and provide robust and reproducible results.Product DescriptionAll-in-One™First-Strand cDNASynthesis KitReverse transcribe mRNA into first–stand cDNAAll-in-One™qPCR PrimersValidated,gene-specific primers ensure specificity and sensitivity (human,mouse and rat)ExProfile™Gene qPCR Arrays High-throughput or focused group profiling of gene expression All-in-One™miRNA First-StrandcDNA Synthesis KitReverse transcribe miRNA into first–stand cDNAAll-in-One™miRNA qRT-PCR Detection Kits SYBR®Green-based detection kit accurately quantifies miRNA expressionAll-in-One™miRNA qPCR Primers Validated human,mouse,rat miRNA primers for robust,reproducible and reliable quantitation of miRNA activitymiProfile™miRNA qPCR Arrays High-throughput or focused group profiling of miRNA expression RNAzol®RT RNA Isolation Reagent Easy isolation of mRNA,microRNA or total RNAIII.Contents and StorageContents and storage recommendations for the All-in-One TM qPCR Mix are provided in the following table. Cat.Nos.QP001,QP002,QP004,and QP005Contents Quantity Storage temperature/conditions2×All-in-One TM qPCR Mix 2×1ml3×(2×1ml)5×(2×1ml)20×(2×1ml)–20°C(Stable for at least12months)Alternatively,the solution can also bestored at–80°C in aliquots.Avoidrepeated freezing/thawing.ROX Reference Dye (30μΜ)1×80µl3×80µl5×80µl20×80µl–20°C(Stable for at least12months)Alternatively,the solution can also bestored at–80°C in aliquots.Avoidrepeated freezing/thawing.IV.PreparationWearing a lab coat,disposable gloves and protective goggles are recommended when handling chemicals.IMPORTANT NOTES:1.When using the All-in-One qPCR Mix with miProfile miRNA qPCR Arrays and All-in-One miRNAFirst-Strand cDNA Synthesis Kit for miRNA expression profiling,please follow the miProfile miRNA qPCR array user manual for the complete instruction.2.Store the kit at–20°C.Avoid storage or leaving reagents at4°C or room temperature.Avoid lightexposure at all times.3.Mix reagents thoroughly by gently inverting tubes several times avoiding bubbles and then brieflycentrifuge before use.4.Prepare the reaction mix with PCR grade water.5.Strictly follow standard procedures for PCR to avoid nucleic acid contamination and non-specificamplification.6.Read all procedures before setting up the PCR reactionV.Procedure1.Thaw the2×All-in-One TM qPCR Mix and ROX Reference Dye as needed.2.Prepare the PCR reaction mix on ice.See the example below.Reagent Volume Final concentration2×All-in-One TM qPCR Mix a10μl1×PCR forward primer(2µM)b2µl0.2µM cPCR reverse primer(2µM)2µl0.2µMTemplate d2μlROX Reference Dye e(30μΜ)ifneeded0.4-0.1μl600nM-150nMWater(double distilled)■Not using ROX Reference Dye4μl■Using ROX Reference Dye3.6-3.9μlTotal20μle the2×All-in-One TM qPCR Mix as half of the total reaction volume and adjust other reagentsaccordingly.If the total reaction volume is changed,maintain each component in the proper proportion. b.Primers are important considerations to ensure success with real-time PCR.All-in-One TM human,mouseand rat primer sets from GeneCopoeia have been validated to provide specific and sensitive amplification even with low copy number genes.For designing your own primers,you may wish to use Oligo primer analysis software(Molecular Biology Insights)or Primer Premier software(Premier Biosoft International).c.Primer concentration should be in the range of0.2to0.6µM.In general,a PCR reaction using0.2µMprimers produces good results.If the PCR efficiency is low,consider increasing primer concentration.However,keep in mind that non-specific PCR products may also increase with increased primer concentration.d.Generally,the amount of DNA template should be less than100ng.Because different templates containvarying copies of a target gene,it may be necessary to perform a gradient dilution to determine the optimal amount of DNA template to use.If reverse transcript cDNA is used as template,dilute before use.Do not add more than5%of the original cDNA solution volume to the total qPCR reaction solution.e.ROX Reference Dye is added only for qPCR instruments that require ROX for calibration.ROXReference Dye provides an internal reference to which the reporter-dye signal can be normalized during data analysis.Normalization is necessary to correct for fluorescence fluctuations due to changes in concentration or volume.Adjust the ROX Reference Dye to optimal concentration according to different qPCR instruments.Instrument ROX per20µl PCR Reaction Final Concentration BioRad iCycler，MyiQ，iQ5，CFX-96，CFX-384，Eppendorf Mastercyclerrealplex，Roche LightCycler480，LightCycler2.0None No ROXABI PRISM7000/7300/7700/7900HTand7900HTFast,ABI Step One,ABI Step One Plus0.4µl（0.2-0.4µl）600nM（300-600nM）ABI7500，7500Fast,ABI Viia7，Stratagene Mx3000P，Mx3005P,Mx4000，0.1µl（0.02-0.1µl）150nM（30-150nM）For other instruments which need calibration of ROX but have not been listed out in the table,please optimize the concentration of ROX according to the guide line of specific instrument.3.Mix the PCR reaction mix sufficiently and add to the PCR reaction tubes.4.Briefly centrifuge to make sure all the reagents are at the bottom of the reaction tubes.5.The following three-step method for programming the PCR reaction is recommended:Cycles Steps Temperature Time Detection 1Initial denaturation95°C10min No40Denaturation95°C10sec No Annealing55°C~60°C20sec No Extension72°C15sec YesNotesi.When using SYBR Green dye to monitor the qPCR reaction,a melting curve analysis should beperformed immediately at the end of cycling.(example adapted from the iQ5real-time PCRdetection system from Bio-Rad):Temperature range Heating rate Constant temperature Detection 72–95°C0.5°C/unit time6sec/unit time Yes25°C30sec NoThe conditions for your instrument may differ,consult the documentation of your qPCR instrument for instructions.ii.The DNA polymerase used in the2×All-in-One TM qPCR Mix is a special chemically modified hot-start enzyme.Incubation for10minutes at95°C will sufficiently activate the enzyme.iii.The actual annealing temperature should be adjusted around the primer melting temperature ranging from55°C~60°C.However,the optimal annealing temperature may be outside of thisrange.Adjust the temperature according to actual reaction conditionsiv.The optimal fragment length to use for amplification during real-time PCR is in the range of80-150bp.However,fragment lengths up to300bp are possible.v.The main condition for the above reaction are referred to in the iQ5qPCR instrument manual from Bio-Rad.If a qPCR instrument from another commercial source is used,please reference theinstrument manual and adjust the extention time and melting curve conditions accordingly.VI.ExampleObjective:The amplification efficiency and detection sensitivity of the2×All-in-One TM qPCR Mix are assessed by standard curves made by gradient dilution of plasmid DNA.The target fragment is102bp.Equipment:iQ5instrument(Bio-Rad Laboratories)Procedure:1.The plasmid is serially diluted to6concentrations ranging from105to1molecule/μl.2.PCR reaction mix preparation(on ice)Reagent components Volume2×All-in-One qPCR Mix10µlPCR forward primer(2µM)2µlPCR reverse primer(2µM)2µlddH201µlTotal15µl3.Mix the above reagents sufficiently.Aliquot to PCR tubes after a brief centrifugation.4.Add5μl of the diluted plasmid template to each PCR e5μl ddH2O as a negative control.5.Program the PCR reaction and corresponding reading conditions of the melting curve:Cycles Steps Temperature Time Detection1Initial denaturation95°C10min No45Denaturation95°C10sec No Annealing60°C20sec No Extension72°C15sec Yes Melting curve reading72°C~95°CHeating Rate0.5°C/6secYes Cooling25°C30sec No6.Analyze the amplification and corresponding melting curves after the qPCR experiment:Amplification curves of serially diluted plasmid DNA Peak values of amplified products in melting curves.7.Construct a standard curve using the Ct values from each amplification curve:Picture of a standard curve8.Conclusion:The peak values from the amplification and melting curves show that as low as5molecules can be detected when using plasmid DNA as a template and that there is only a single amplified product,showing that very high sensitivity can be attained using the All-in-One TM qPCR Mix.At the same time,high amplification efficiency is also shown by the good linear relationship among each concentration of serially diluted plasmid.VII.Trouble Shooting GuidePoor precision or failed qPCR reactions ∙Make sure the initial denature time was set as10min,sufficiently activating of the hot-start polymerase could avoid non-specific amplification and production of primer-dimers.∙The fluorescence detection temperature may not be appropriate.Adjust accordingly.∙The set up position for reaction samples in the real-time PCR instrument may not be right.Adjust accordingly.∙PCR cycle conditions,primer concentration and primer sequences may not be appropriate.Adjust the primer concentration and annealing temperature.If this does not work,redesign the primers.∙The template sample purity may not be adequate.Purify the template sample by phenol/chloroform extraction and ethanol precipitation.If the samples are reverse transcribed cDNA,set up the qPCR reaction with a diluted sample as other concentrated reagents in the RT reaction mixture may be interfering with the qPCR.∙Try to use 3.0%agarose gel electrophoresis to check the qPCR products.Check the purity of the primers by electrophoresis or use PAGE-purified primers if the bands are diffused.One may also use phenol/chloroform extraction and ethanol precipitation methods to treat the primers before the experiment.Abnormal meltingcurvesSignal in the blank(No Template Control)sample∙There may be contamination of the positive samples in the qPCR reaction system if the T m of the melting curve of the blank control is the same as the positive control.Eliminate sample application error first.If the situation still persists,replace the PCR grade water and/or primers and/or use a new2×All-in-One TM qPCR Mix.∙If the T m of the melting curve of the blank control is lower than the positive control,the qPCR reaction may have produced nonspecific amplification such as primer-dimers.Prepare the qPCR reaction mix on ice and increase the temperature of fluorescence detection.If this does not work,redesign the primers.Double peaks and multiple peaks in the melting curve of the positive control∙In the absence of other primers present in the reaction,double or multiple peaks in the melting curve of the positive control indicate that the qPCR reaction produced nonspecific amplification fragments.Prepare the qPCR reaction mix on ice;optimize the qPCR reaction conditions,for example,by increasing the annealing temperature, decreasing the primer concentration or increasing the fluorescence detection temperature(not more than the T m value of the expected product).If this does not work,redesign the forward primer.No peaks or abnormal peaks in the melting curve(or the amplification curves)of the positive control∙Adjust the ROX Dye to optimized concentration according to instrument.No signal(Ct)or late appearing signal ∙Not enough PCR cycles.For good sensitivity,one should generally set up more than35PCR cycles,but more than45cycles may result in too much background signal.∙The amount of template used may not be enough or the template may be e the highest concentration possible of diluted template samples to set up the qPCR.At the same time,avoid freezing and thawing the samples repeatedly.∙The amplification efficiency is low and the qPCR reaction conditions are not optimal.Redesign the primers and optimize the reaction conditions.VIII.Limited Use License and WarrantyLimited Use LicenseFollowing terms and conditions apply to use of all OmicsLink™ORF Expression Clones in all lentiviral vectors and Packaging Kit(theProduct).If the terms and conditions are not acceptable,the Product in its entirety must be returned to GeneCopoeia within5calendar days.A limited End-User license is granted to the purchaser of the Product.The Product shall be used by the purchaser for internal researchpurposes only.The Product is expressly not designed,intended,or warranted for use in humans or for therapeutic or diagnostic use.TheProduct must not be resold,repackaged or modified for resale,or used to manufacture commercial products without prior written consentfrom GeneCopoeia.This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetice of any part of the Product constitutes acceptance of the above terms.Limited WarrantyGeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet.If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications,GeneCopoeia will replace the Product.In the event a replacement cannot be provided,GeneCopoeia will provide the purchaser with a refund.This limited warranty shall not extend to anyone other than the original purchaser of the Product.Notice of nonconforming products must be made to GeneCopoeia within30days of receipt of the Product.GeneCopoeia’s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price.GeneCopoeia’s liability does not extend to any damages arising from use or improper use of the Product,or losses associated with the use of additional materials or reagents.This limited warranty is the sole and exclusive warranty.GeneCopoeia does not provide any other warranties of any kind,expressed or implied,including the merchantability or fitness of the Product for a particular purpose.GeneCopoeia is committed to providing our customers with high-quality products.If you should have any questions or concerns about anyGeneCopoeia products,please contact us at301-762-0888.©2016,GeneCopoeia,Inc.GeneCopoeia,Inc.9620Medical Center Drive,#101Rockville,MD20850Tel:301-762-0888Fax:301-762-3888Email:***********************Web:GeneCopoeia Products are for Research Use Only Copyright©2016GeneCopoeia,Inc. Trademarks:GeneCopoeia™,All-in-One™,ExProfile™,miProfile™(GeneCopoeia Inc.);RNAzol®(Molecular Research Center,Inc.);SYBR®(Molecular Probes);iQ™5(Bio-Rad);ROX®(Invitrogen).QP001020216。
Extract-N-Amp Tissue PCR Kit 产品说明书
Product InformationExtract-N-Amp™ Tissue PCR KitXNAT2, XNAT2RProduct DescriptionThe Extract-N-Amp™ Tissue PCR Kit for direct PCR contains the reagents needed to rapidly extract and amplify genomic DNA from mouse tails and other animal tissues, buccal swabs, hair shafts, and saliva. Briefly, the DNA is released from the starting material by incubating the sample with a mixture of the Extraction Solution and the Tissue Preparation Solution at room temperature for 10 minutes. There is no need for mechanical disruption, organic extraction, column purification, or precipitation of the DNA.After adding Neutralization Solution B, the extract is ready for PCR. An aliquot of the neutralized extract is then combined with the Extract-N-Amp™ PCR Reaction Mix and user-provided PCR primers to amplify target DNA. The Extract-N-Amp™ PCR Reaction Mix is a 2X ready mix containing buffer, salts, dNTPs, and Taq polymerase. It is optimized specifically for use with the extraction reagents. It also contains the JumpStart Taq antibody for hot start PCR to enhance specificity but does not contain the inert red dye found in the REDExtract-N-Amp™ PCR Reaction Mix.Reagents Provided Cat. No. XNAT2 100 Preps,100 PCRsXNAT2R 1000 Preps, 1000 PCRsExtraction SolutionE7526 24 mL 240 mL Tissue Preparation Solution T3073 3 mL 30 mL Neutralization Solution BN391024 mL240 mLExtract-N-Amp™ PCR Reaction Mix This is a 2X PCR reaction mix containing buffer, salts, dNTPs, Taq polymerase, and JumpStart™ Taq antibody.E30041.2 mL12 mLReagents and Equipment Required(Not Provided)•Microcentrifuge tubes (1.5 or 2 mL) or multi-well plate for extractions (200 μL minimal well volume) • Small dissecting scissors• Forceps (small to medium in size)• Buccal swab - Sterile foam tipped applicator (Cat. No. WHAWB100032)•Sample collection card - Bloodstain card (Cat. No. WHAWB100014)• Tubes or plate for PCR• Heat block or thermal cycler at 95 °C • PCR Primers (Cat. No. OLIGO) • Thermal cycler•Water, PCR Reagent (Cat. No. W1754)Precautions and DisclaimerThis product is for R&D use only. Not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.StorageThe Extract-N-Amp™ Tissue PCR Kit can be stored at 2 to 8 °C for up to 3 weeks. For long-term storage, greater than 3 weeks, -20 °C is recommended. Do not store in a "frost-free" freezer.ProcedureAll steps are carried out at room temperature unless otherwise noted.DNA Extraction from Mouse Tails, Animal Tissues, Hair, or Saliva1.Pipette 100 μL of Extraction Solution into amicrocentrifuge tube or well of a multi-well plate.Add 25 μL of Tissue Preparation Solution to thetube or well and pipette up and down to mix.Note: If several extractions will be performed,sufficient volumes of Extraction and TissuePreparation Solutions may be pre-mixed in a ratio of 4:1 up to 2 hours before use.2.For fresh or frozen mouse tails: Rinse thescissors and forceps in 70% ethanol prior to useand between different samples. Place a 0.5–1 cm piece of mouse tail tip (cut end down) into thesolution. Mix thoroughly by vortexing or pipetting.Ensure the mouse tail is in solution.Note: For fresh mouse tails, perform extractions within 30 minutes of snipping the tail.For animal tissues: Rinse the scissors or scalpel and forceps in 70% ethanol prior to use andbetween different samples. Place a 2–10 mgpiece of tissue into the solution. Mix thoroughlyby vortexing or pipetting. Ensure the tissue is inthe solution.For hair shafts: Rinse the scissors and forceps in 70% ethanol prior to use and between differentsamples. Trim excess off of the hair shaft leaving the root and place sample (root end down) intosolution. Only one hair shaft, with root, isrequired per extraction.For Saliva: Pipette 10 μL of saliva into thesolution. Mix thoroughly by vortexing or pipetting.For saliva dried on card: Pipette 50 μL of saliva onto collection card and allow the card to dry.Rinse the punch in 70% ethanol prior to use andbetween different samples. Punch a disk(preferably 1/8 inch or 3 mm) out of the cardfrom the area with the dried saliva sample. Place disk into the solution. Tap tube or plate on hardsurface to ensure disk is in solution forincubation period.3.Incubate sample at room temperature for10 minutes.4.Incubate sample at 95 °C for 3 minutes.Note: Tissues will not be completely digested atthe end of the incubations. This is normal and will not affect performance.5.Add 100 μL of Neutralization Solution B to sampleand mix by vortexing.6.Store the neutralized tissue extract at 4 °C oruse immediately in PCR amplification.Note: For long term storage, remove theundigested tissue or transfer the extracts tonew tubes or wells. Extracts may now be storedat 4 °C for at least 6 months without notable loss in most cases.DNA Extraction for Buccal Swabs1.Collect buccal cells on swab and allow theswab to dry. Drying time is approximately10 to 15 minutes.Note: Due to the low volume of solution used for DNA extraction, a foam tipped swab should beused. Swabs with fibrous tips, such as cotton orDacron®, should be avoided because the solution cannot be recovered efficiently.2.Pipette 200 μL of Extraction Solution into amicrocentrifuge tube. Add 25 μL of TissuePreparation Solution to the tube and pipette upand down to mix.Note: If several extractions will be performed,sufficient volumes of Extraction and TissuePreparation Solutions may be pre-mixed ina ratio of 8:1 up to 2 hours before use.3.Place dried buccal swab into solution and incubateat room temperature for 1 minute.4.Twirl swab in solution 10 times and then removeexcess solution from the swab into the tube bytwirling swab firmly against the side of the tube.Discard the swab. Close the tube andvortex briefly.5.Incubate sample at room temperature for10 minutes.6.Incubate sample at 95 °C for 3 minutes.7.Add 200 μL of Neutralization Solution B to sampleand mix by vortexing.8.Store the neutralized extract at 4 °C or useimmediately in PCR. Continue to PCRamplification.Note: Extracts may be stored at 4 °C for at least6 months without notable loss in most cases. PCR AmplificationThe Extract-N-Amp™ PCR Reaction Mix contains JumpStart™ Taq antibody for specific hot start amplification. Therefore, PCR mixtures can be assembled at room temperature without premature Taq DNA polymerase activity.Typical final primer concentrations are approximately 0.4 μM each. The optimal primer concentration and cycling parameters will depend on the system being used.1.Add the following reagents to a thin-walled PCRmicrocentrifuge tube or plate:Reagent VolumeWater, PCR grade VariableExtract-N-Amp™ PCRreaction mix 10 μLForward primer VariableReverse primer VariableTissue extract 4 μL*Total volume 20 μL*The Extract-N-Amp™ PCR Reaction Mix isformulated to compensate for components in the Extraction, Tissue Preparation, and Neutralization Solutions. If less than 4 µL of tissue extract isadded to the PCR reaction volume, use a 50:50mixture of Extraction and Neutralization BSolutions to bring the volume of tissue extract upto 4 μL.2.Mix gently.3.For thermal cyclers without a heated lid, add20 μL of mineral oil on top of the mixture in eachtube to prevent evaporation.4.Perform thermal cycling. The amplificationparameters should be optimized for individualprimers, template, and thermal cycler.Common cycling parameters:Step Temperature Time Cycles InitialDenaturation 94 °C 3 minutes 1 Denaturation 94 °C 30 seconds Annealing 45 to 68 °C 30 seconds 30-35 Extension 72 °C 1-2 minutes(1 min/kb)FinalExtension 72 °C 10 minutes 1 Hold 4 °C Indefinitely5.The amplified DNA can be loaded onto an agarosegel after the PCR is completed with the addition ofa separate loading buffer/tracking dye such as GelLoading Solution, Cat. No. G2526.Note: PCR products can be purified, if desired, fordownstream applications such as sequencing withthe GenElute PCR Clean-Up Kit, Cat. No.NA1020.Troubleshooting GuideProblem Cause SolutionLittle or no PCR product is detected. PCR reaction may beinhibited due tocontaminants in thetissue extract.Dilute the tissue extract with a 50:50 mix of Extractionand Neutralization Solutions. To test for inhibition, includea DNA control and/or spike a known amount of template(100-500 copies) into the PCR along with the tissue extract. Extraction isinsufficient.Incubate samples at 55 °C for 10 minutes instead ofroom temperature.A PCR component maybe missing or degraded.Run a positive control to ensure that componentsare functioning. A checklist is also recommendedwhen assembling reactions.There may be too fewcycles performed. Increase the number of cycles (5-10 additional cycles at a time). The annealingtemperature maybe too high.Decrease the annealing temperature in 2-4 °C increments.The primers may notbe designed optimally.Confirm the accuracy of the sequence information. If theprimers are less than 22 nucleotides long, try to lengthen theprimer to 25-30 nucleotides. If the primer has a GC contentof less than 45%, try to redesign the primer with a GCcontent of 45-60%.The extension timemay be too short.Increase the extension time in 1-minute increments, especiallyfor long templates.Target templateis difficult.In most cases, inherently difficult targets are due to unusuallyhigh GC content and/or secondary structure. Betaine, Cat. No.B0300, has been reported to help amplification of high GCcontent templates at a concentration of 1.0-1.7 M.Multiple products JumpStart™ Taqantibody is notworking correctly.Do not use DMSO or formamide with Extract-N-Amp™ PCRReaction Mix. It can interfere with the enzyme-antibodycomplex. Other cosolvents, solutes (e.g., salts), and extremesin pH or other reaction conditions may reduce the affinity ofthe JumpStart™ Taq antibody for Taq polymerase and therebycompromise its effectiveness.TouchdownPCR maybe needed.“Touchdown” PCR significantly improves the specificity of manyPCR reactions in various applications. Touchdown PCR involvesusing an annealing/extension temperature that is higher thanthe TM of the primers during the initial PCR cycles. Theannealing/extension temperature is then reduced to the primerTM for the remaining PCR cycles. The change can be performedin a single step or in increments over several cycles.Negative control shows a PCR product or “false positive” result. Reagents arecontaminated.Include a reagent blank without DNA template be included asa control in every PCR run to determine if the reagents used inextraction or PCR are contaminated with a template froma previous reaction.Tissue is not digested after incubations. Tissue is not expectedto be completelydigested.The REDExtract-N-Amp™ Tissue PCR Kit does not require thetissue to be completely digested. Sufficient DNA is released forPCR without completely digesting the tissue.Buccal swab absorbed all the solution. The recommended typeof swab was not used.Due to the low volume of solution used for DNA extraction, afoam tipped swab should be used. Swabs with fibrous tips, suchas cotton or Dacron®, should be avoided because the solutioncannot be recovered efficiently.References1.Dieffenbach, C.W., and Dveksler, G.S. (Eds.), PCRPrimer: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1995).2.Don, R.H. et al., ‘Touchdown' PCR to circumventspurious priming during gene amplification.Nucleic Acids Res., 19, 4008 (1991).3.Erlich, H.A. (Ed.), PCR Technology: Principles andApplications for DNA Amplification, StocktonPress, New York (1989).4.Griffin, H.G., and Griffin, A.M. (Eds.), PCRTechnology: Current Innovations, CRC Press,Boca Raton, FL (1994).5.Innis, M.A., et al., (Eds.), PCR Strategies,Academic Press, New York (1995).6.Innis, M., et al., (Eds.), PCR Protocols: A Guide toMethods and Applications, Academic Press, SanDiego, California (1990).7.McPherson, M.J. et al., (Eds.), PCR 2: A PracticalApproach, IRL Press, New York (1995).8.Newton, C.R. (Ed.), PCR: Essential Data, JohnWiley & Sons, New York (1995).9.Roux, K.H. Optimization and troubleshooting inPCR. PCR Methods Appl., 4, 5185-5194 (1995).10.Saiki, R., PCR Technology: Principles andApplications for DNA Amplification, Stockton, New York (1989). Product OrderingOrder products online at Related Products Cat. No.Ethanol E7148; E7023; 459836 Forceps,micro-dissecting F4267PCR Marker P9577PCR microtubes Z374873; Z374962;Z374881PCR multi-well plates Z374903Precast Agarose Gels P6097Sealing mats & tapes Z374938; A2350TBE Buffer T4415, T6400, T9525The life science business of Merck operatesas MilliporeSigma in the U.S. and Canada.Merck, Extract-N-Amp, REDExtract-N-Amp, JumpStart, GenElute and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are theproperty of their respective owners. Detailed information on trademarks is available via publicly accessible resources.NoticeWe provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose. The information in this document is subject to change without notice and should not be construed as a commitment by the manufacturing or selling entity, or an affiliate. We assume no responsibility for any errors that may appear in this document. Technical AssistanceVisit the tech service page at/techservice.Terms and Conditions of SaleWarranty, use restrictions, and other conditions of sale may be found at /terms. Contact InformationFor the location of the office nearest you, go to /offices.。
抗核抗体检测试剂盒(磁条码免疫荧光法)产品技术要求丽珠
1性能指标
1.1外观
试剂盒各组份应齐全、完整，标签清晰，易识别；液体无渗漏。
1.2准确度
对准确度参考品进行检测，双链DNA（dsDNA）IgG抗体的相对偏差应在±20%范围内。
1.3最低检测限
对检测限参考品进行检测，dsDNA IgG抗体的检测限应不高于10 IU/mL；其它定性指标对应的各指标的L1应为阳性、L2应为阳性或阴性、L3应为阴性。
1.4线性
dsDNA IgG抗体在10~ 600 IU/mL范围内，其线性相关系数（r）r2应不小于0.950。
1.5阴性参考品符合率
对10份阴性参考品进行检测，对应指标的阴性参考品符合率应为100%。
1.6阳性参考品符合率
对12份阳性参考品进行检测，对应指标的阳性参考品符合率应为100%。
1.7重复性
对重复性参考品检测10次，同一指标的变异系数（CV）应不大于15%。
1.8批间差
用三个批号试剂盒检测同一份重复性参考品，批间差应不大于20%。
1.9dsDNA 校准品均匀性
1.9.1瓶内均匀性
校准品的瓶内均匀性（变异系数，CV）应不大于15%。
1.9.2瓶间均匀性
校准品的瓶间均匀性（变异系数，CV）应不大于15%。
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PCNA核内参抗体，抗PCNA单抗，PCNA小鼠源单克隆抗体
Anti-PCNA Mouse Monoclonal Antibody （ 1D7）
增殖细胞核抗原（Proliferating Cell Nuclear Antigen简称PCNA）由Miyachi等于1978年在 SLE（系统性红斑狼疮）患者的血清中首次发现并命名，因其只存在于正常增殖细胞及肿瘤细胞内而得名。
PCNA是一种分子量为36KD的蛋白质，在细胞核内合成，并存在于细胞核内，为DNA聚合酶的辅助蛋白。
由于其在细胞核内稳定表达，因此其抗体被广泛应用于细胞核蛋白的内参抗体。
如何选择合适的细胞核内参抗体？
常用的细胞总蛋白质内参有GAPDH和细胞骨架蛋白β-Actin或β-Tubulin等。
对于一些植物的样本，则需要特别的植物Actin蛋白作为内参。
当实验样品中只是核蛋白，而不是细胞总蛋白提取液时，可以用组蛋白H（Histone H），或者增殖细胞核抗原（PCNA）等为核内参抗体。
除了这些，其它常见的核蛋白内参还有K70, K80, Lamin A和B，在一些文献报道中，Erk2、TATA binding protein（TBP）以及c-Jun、c-Fos等都有使用。
但是需要注意的问题是核蛋白内参的选择需要考虑实际的试验环境，比如在涉及细胞增殖相关试验中，c-Jun由于自身表达变化就不适合做内参；而在凋亡实验时，TBP、Lamin等也不适合作为内参。
应用类型：WB（1:1000-1:5,000）、IHC（1:100-1:500）
Fig.1.Immunohistochemistry staining (1:100) of Human breast cancer tissue paraffin-embedded with Anti-PCNA mouse monoclonal antibody (1D7).
Fig.2.Western blot analysis (1:2,000) of PCNA expression in Rat brain (lane A), A549 cell (lane B), NIH 3T3 cell (lane C) and 293T cell (lane D) with Anti-PCNA mouse monoclonal antibody (1D7).
免疫原：KLH偶联的PCNA部分多肽序列
来源宿主：小鼠
反应性：该PCNA核内参抗体可识别人、小鼠、大鼠等种属的PCNA蛋白。
保存建议：在发货后能在-20℃保存1年。
为最大限度的避免损失，请在打开管盖之前融化抗体并离心。
我们建议使用前分装以避免反复冻融，分装后可在4℃保存1个月。
背景资料：增殖细胞核抗原（ Proliferating Cell Nuclear Antigen简称PCNA）由Miyachi等于1978年在 SLE（系统性红斑狼疮）患者的血清中首次发现并命名，因其只存在于正常增殖细胞及肿瘤细胞内而得名。
PCNA是一种分子量为36KD的蛋白质，在细胞核内合成，并存在于细胞核内，为DNA聚合酶的辅助蛋白。
由于其在细胞核内稳定表达，因此其抗体被广泛应用于细胞核蛋白的内参抗体。