Cationic liposomes are traditionally used for the delivery of genetic materials such as various types of DNA (pDNA, cDNA, CpG DNA, oligonucleotide, antisense oligonucleotide, etc.), various types of RNA such as (siRNA, mRNA, etc.) and nucleic acid mimics (NAMs). The encapsulation of DNA into the conventional neutral charged PC based liposomes can be a technical problem mainly due to the plasmid size. Due to this problem in late 80s, the liposomes composed of cationic lipids and PE have been developed. The idea was to neutralize the negative charge of pDNA with positive charge of cationic lipids in order to capture more plasmid efficiently mainly due to electrostatic interaction and deliver them into the cells. Generally, the procedure is simply based on mixing the cationic liposomes with DNA or RNA and adding them to the cells. This results in the formulations of aggregates.
In order to design a proper cationic lipid for gene delivery, two approaches have been used for the cationic lipid synthesis: 1) cholesterol-based design such as DC-Cholesterol and GL-67 lipids, and 2) non-cholesterol-based designs such as DOTAB, DDAB and DOTMA. To successfully transfer the gene in vitro using liposomes, some consideration should be taken into account: i) the ability of binding and packing DNA/RNA in liposomes; ii) the interaction of the packaged DNA/RNA to the cell surface; iii) the efficiency of the internalization of DNA/RNA; iv) the intracellular DNA-release from endosomes in case of endocytosis involvement; v) the transgenic expression level in cell nuclei. pH-sensitive liposomes have been designed based on their tendency to release their content in the acidic condition. The primary concept was based on viruses that fuse with the endosomal membrane by means of a protein at pH 5-6, delivering their genetic material to the cytosol before reaching the lysosomes. Typically, a pH-sensitive liposome consists of dioleoylphosphatidylethanolamine (DOPE). Since phosphatidylethanolamine (PE) changes in acidic conditions, it is believed to act as a membrane fusion promoter. The effectiveness of the interaction between liposomes and cells is highly dependent on the liposome compositions. Liposomes are captured by various endocytosic processes, and the efficiency depends on the cell type and liposome size. Liposomes of various sizes and charges can attach to the macrophages and neutrophils through active phagocytosis. After attachment of the liposome to the cell surface, the internalization into the endosomes occurs due to a more acidic pH (6.50) at early endosomes. The liposomes are transferred to the last endosome with more acidic pH (5.5-6.0) by maturation or vesicular fusion, which takes 10-15 min. Twenty minutes (or more) after uptake, the contents are delivered to the lysosome with pH 5.0 or less. Lysosomes are the main degrading and last endocytotic section in the endocytotic pathway, in where pH-insensitive liposomes are accumulated and degraded. However, after penetration of pH-sensitive liposomes into cells, the accumulation and degradation do not occur.
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探针
ddH2O36.25ul
buffer(瓶3)5ul
核苷酸混合液(含DIG-dUTP)(瓶2)2.5ul
dNTPStockSolution(瓶4)2.5ul
引物F1ul(10pmol/ul)
R1ul(10pmol/ul)
酶(瓶1)0.75ul
DNA1ul(100pg)
程序:35个循环,退火温度55度。
第一次通过探针标记得到了相应的PCR标记产物,亮度和对照相当,片段大小比标记的探针模板大,证明整个过程应该没有问题。
但一个月后重新用相同的体系,相同的程序,只是探针模板的量不同却什么都扩不出来。不知道问题出在哪了!
按照药品管理的用于血源筛查的体外诊断试剂和采用放射性核素标记的体外诊断试剂,不属于本办法管理范围。
第十七条 根据产品风险程度由低到高,体外诊断试剂分为第一类、第二类、第三类产品。
(一)第一类产品
1.微生物培养基(不用于微生物鉴别和药敏试验);
2.样本处理用产品,如溶血剂、稀释液、染色液等。
(二)第二类产品
除已明确为第一类、第三类的产品,其他为第二类产品,主要包括:
1.用于蛋白质检测的试剂;
2.用于糖类检测的试剂;
3.用于激素检测的试剂;
4.用于酶类检测的试剂;
5.用于酯类检测的试剂;
6.用于维生素检测的试剂;
7.用于无机离子检测的试剂;
8.用于药物及药物代谢物检测的试剂;
9.用于自身抗体检测的试剂;
10.用于微生物鉴别或者药敏试验的试剂;
11.用于其他生理、生化或者免疫功能指标检测的试剂。
(三)第三类产品
1.与致病性病原体抗原、抗体以及核酸等检测相关的试剂;
2.与血型、组织配型相关的试剂;
3.与人类基因检测相关的试剂;
4.与遗传性疾病相关的试剂;
5.与麻醉药品、精神药品、医疗用毒性药品检测相关的试剂;
6.与治疗药物作用靶点检测相关的试剂;
7.与肿瘤标志物检测相关的试剂;
8.与变态反应(过敏原)相关的试剂。
生物偶联即将两种分子结合在一起而形成一个杂合的偶联物(hybridconjugate),这些偶联结合物(如标记抗体)在世界各大医院和科研机构被广泛使用。一抗标记试剂系列产品极大程度的简化了结合标记流程,是目前世界上操作最简便的抗体标记试剂盒。
·快速:手工操作时间仅30秒·目前世界上操作最简便的抗体标记试剂盒·无需脱盐、透析等步骤·抗体回收率达100%·可提供数十种标记物·可用于WB,IHC,ELISA,免疫荧光、流式细胞术以及荧光共振能量转移检测等·经济实惠,规格多样·可标记ug-mg级抗体量试剂盒组成:
1)1或3(以规格而定)玻管Lightening-Link?Mix混合物。
2)1管LL-X(X为标记物,如PE或HRP等)修饰试剂。
3)1管LL-X萃灭试剂。
操作步骤:
1)在每10ul抗体样品中加入1ul的LL-Modifier修饰试剂,轻轻混匀。
2)打开Ligtening-Link混合物管盖,吸取步骤1已混好的抗体样品至管内冻干混合物中,用吸液器轻轻吸打1-2次,使粉末完全溶解。
3)盖上玻璃瓶盖,室温(20-25°C)避光孵育3小时,或过夜。*
4)孵育3小时后,按每10ul抗体的量加入1ulLL-淬灭试剂,静置30分钟后即可使用。
*不同的标记物,操作步骤略有不同。(操作步骤供参考,以英文原版说明书为准。)
标记结合物的保存:
对于任何新结合物,推荐保持于4°C条件下。如需长期保存,可添加保护剂。
缓冲液及添加剂兼容性:
A.与无胺基缓冲液兼容,包括MES,MOPS,HEPES(浓度10-50mM,pH范围6.5-8.5),LL-Modifier修饰剂提供结合反应的最佳条件。
B.常见的盐(如氯化钠)、螯合剂(EDTA)、糖对偶联反应无影响。
C.叠氮钠(0.02%-0.1%)对结合反应有轻微影响,叠氮钠不可用于HRP标记,其为后者的抑制剂。
D.甘油(upto50%)对结合效率无影响。
E.若无胺基的缓冲液浓度相对较高,pH值在6.6-8.5范围外,则需要加入更多的LL-midifier修饰剂(/10ul抗体),以此来调整pH值。最佳的pH范围为7.3-7.6左右。
F.避免使用含亲核剂的缓冲液,避免还原剂如DTT和2-巯基乙醇,乙醇胺等。
G.浓度达到或稍低于20mM的Tris对结合效率有轻微的影响。
Westernblottingdatacomparingconjugatedandunconjugatedantibodies
货号中文品名英文品名规格AZK0001AColorlockALS磷检测试剂盒PiColorlockALS625/1560assaysAZK0001BColorlockALS磷检测试剂盒PiColorlockALS2500/6250assaysAZK0002AColorlockGold磷检测试剂盒PiColorlockGold625/1560assaysAZK0002BColorlockGold磷检测试剂盒PiColorlockGold2500/6250assaysAZK0003APi结合树脂PiBind5gAZK0004AATP琼脂糖Low(1-2umol/ml)ATPagaroseLow(1-2umol/ml)2mlAZK0004BATP琼脂糖Low(1-2umol/ml)ATPagaroseLow(1-2umol/ml)5mlAZK0005AATP琼脂糖High(>6umol/ml)GTPagaroseHigh(>6umol/ml)2mlAZK0006AATP琼脂糖High(1-2umol/ml)GTPagaroseHigh(>6umol/ml)5mlAZK0007AATP琼脂糖High(8-12umol/ml)ATPagaroseHigh(8-12umol/ml)2mlAZK0008AATP琼脂糖High(8-12umol/ml)ATPagaroseHigh(8-12umol/ml)5mlAZK0009A控制树脂ControlResin2mlAZK0009B控制树脂ControlResin5mlAZK0010AATPase酶检测试剂盒ATPaseassaysystem192assaysAZK0010BATPase酶检测试剂盒ATPaseassaysystem480assaysAZK0010CATPase酶检测试剂盒ATPaseassaysystem5x480assaysAZK0011AGTPase酶检测试剂盒GTPaseassaysystem192assaysAZK0011BGTPase酶检测试剂盒GTPaseassaysystem480assaysAZK0011CGTPase酶检测试剂盒GTPaseassaysystem5x480assaysAZK0013AAP碱性磷酸酶抗体标记试剂盒Lightning-LinkAPconjugationkit3x10μgAPAZK0013BAP碱性磷酸酶抗体标记试剂盒Lightning-LinkAPconjugationkit1x100μgAPAZK0013CAP碱性磷酸酶抗体标记试剂盒Lightning-LinkAPconjugationkit3x100μgAPAZK0013DAP碱性磷酸酶抗体标记试剂盒Lightning-LinkAPconjugationkit1mgAPAZK0014AGOx葡糖氧化酶标记试剂盒Lightning-LinkGOxconjugationkit3x10μgAZK0014BGOx葡糖氧化酶标记试剂盒Lightning-LinkGOxconjugationkit3x100μgAZK0014CGOx葡糖氧化酶标记试剂盒Lightning-LinkGOxconjugationkit1x1mgAZK0015AR-PE(PE)R-藻红蛋白标记试剂盒Lightning-LinkR-Phycoerythrin(R-PE)3x10μgPEAZK0015BR-PE(PE)R-藻红蛋白标记试剂盒Lightning-LinkR-Phycoerythrin(R-PE)1x100μgPEAZK0015CR-PE(PE)R-藻红蛋白标记试剂盒Lightning-LinkR-Phycoerythrin(R-PE)3x100μgPEAZK0015DR-PE(PE)R-藻红蛋白标记试剂盒Lightning-LinkR-Phycoerythrin(R-PE)1mgPEAZK0016AB-PEB-藻红蛋白标记试剂盒Lightning-LinkB-Phycoerythrin(B-PE)1x100μgBPEAZK0016BB-PEB-藻红蛋白标记试剂盒Lightning-LinkB-Phycoerythrin(B-PE)3x100μgBPEAZK0016CB-PEB-藻红蛋白标记试剂盒Lightning-LinkB-Phycoerythrin(B-PE)1mgBPEAZK0017AAPC别藻蓝蛋白标记试剂盒Lightning-LinkAllophycocyanin(APC)3x10μgAPCAZK0017BAPC别藻蓝蛋白标记试剂盒Lightning-LinkAllophycocyanin(APC)1x100μgAPCAZK0017CAPC别藻蓝蛋白标记试剂盒Lightning-LinkAllophycocyanin(APC)3x100μgAPCAZK0017DAPC别藻蓝蛋白标记试剂盒Lightning-LinkAllophycocyanin(APC)1mgAPCAZK0018APerCP标记试剂盒Lightning-LinkPerCP3x10ugAZK0018BPerCP标记试剂盒Lightning-LinkPerCP1x100ugAZK0018CPerCP标记试剂盒Lightning-LinkPerCP3x100ugAZK0018DPerCP标记试剂盒Lightning-LinkPerCP1x1mgAZK0019A荧光素标记试剂盒Lightning-LinkFluorescein3reactions(eachupto20ug)AZK0019B荧光素标记试剂盒Lightning-LinkFluorescein3reactions(eachupto200ug)AZK0019C荧光素标记试剂盒Lightning-LinkFluorescein1reaction(upto2mg)AZK0020A罗丹明标记试剂盒Lightning-LinkRhodamine3reactions(eachupto20ug)AZK0020B罗丹明标记试剂盒Lightning-LinkRhodamine3reactions(eachupto200ug)AZK0020C罗丹明标记试剂盒Lightning-LinkRhodamine1reaction(upto2mg)AZK0021AAMCA标记试剂盒Lightning-LinkAMCA3reactions(eachupto20ug)AZK0021BAMCA标记试剂盒Lightning-LinkAMCA3reactions(eachupto200ug)AZK0021CAMCA标记试剂盒Lightning-LinkAMCA1reaction(upto2mg)AZK0022ATexasRed标记试剂盒Lightning-LinkTexasRed3reactions(eachupto20ug)AZK0022BTexasRed标记试剂盒Lightning-LinkTexasRed3reactions(eachupto200ug)AZK0022CTexasRed标记试剂盒Lightning-LinkTexasRed1reaction(upto1mgscale)AZK0023ACy3染料标记试剂盒Lightning-LinkCy33reactions(eachupto20ug)AZK0023BCy3染料标记试剂盒Lightning-LinkCy33reactions(eachupto200ug)AZK0023CCy3染料标记试剂盒Lightning-LinkCy31reaction(upto2mg)AZK0024ACy5染料标记试剂盒Lightning-LinkCy53reactions(eachupto20ug)AZK0024BCy5染料标记试剂盒Lightning-LinkCy53reactions(eachupto200ug)AZK0024CCy5染料标记试剂盒Lightning-LinkCy51reaction(upto2mg)AZK0025ACy5.5染料标记试剂盒Lightning-LinkCy5.53reactions(eachupto200ug)AZK0025BCy5.5染料标记试剂盒Lightning-LinkCy5.51reaction(upto2mg)AZK0026ACy7染料标记试剂盒Lightning-LinkCy73reactions(eachupto20ug)AZK0026BCy7染料标记试剂盒Lightning-LinkCy73reactions(eachupto200ug)AZK0026CCy7染料标记试剂盒Lightning-LinkCy71reaction(upto2mg)AZK0027ACy3.5染料标记试剂盒Lightning-LinkCy3.53reactions(eachupto200ug)AZK0027BCy3.5染料标记试剂盒Lightning-LinkCy3.51reaction(upto2mg)AZK0028AAtto390荧光染料标记试剂盒Lightning-Linkatto3903reactions(eachupto20ug)AZK0028BAtto390荧光染料标记试剂盒Lightning-Linkatto3903reactions(eachupto200ug)AZK0028CAtto390荧光染料标记试剂盒Lightning-Linkatto3901reaction(upto2mg)AZK0029AAtto425荧光染料标记试剂盒Lightning-Linkatto4253reactions(eachupto20ug)AZK0029BAtto425荧光染料标记试剂盒Lightning-Linkatto4253reactions(eachupto200ug)AZK0029CAtto425荧光染料标记试剂盒Lightning-Linkatto4251reaction(upto2mg)AZK0030AAtto465荧光染料标记试剂盒Lightning-Linkatto4653reactions(eachupto20ug)AZK0030BAtto465荧光染料标记试剂盒Lightning-Linkatto4653reactions(eachupto200ug)AZK0030CAtto465荧光染料标记试剂盒Lightning-Linkatto4651reaction(upto2mg)AZK0031AAtto488荧光染料标记试剂盒Lightning-Linkatto4883reactions(eachupto20ug)AZK0031BAtto488荧光染料标记试剂盒Lightning-Linkatto4883reactions(eachupto200ug)AZK0031CAtto488荧光染料标记试剂盒Lightning-Linkatto4881reaction(upto2mg)AZK0032AAtto532荧光染料标记试剂盒Lightning-Linkatto5323reactions(eachupto20ug)AZK0032BAtto532荧光染料标记试剂盒Lightning-Linkatto5323reactions(eachupto200ug)AZK0032CAtto532荧光染料标记试剂盒Lightning-Linkatto5321reaction(upto2mg)AZK0033AAtto565荧光染料标记试剂盒Lightning-Linkatto5653reactions(eachupto20ug)AZK0033BAtto565荧光染料标记试剂盒Lightning-Linkatto5653reactions(eachupto200ug)AZK0033CAtto565荧光染料标记试剂盒Lightning-Linkatto5651reaction(upto2mg)AZK0034AAtto590荧光染料标记试剂盒Lightning-Linkatto5903reactions(eachupto20ug)AZK0034BAtto590荧光染料标记试剂盒Lightning-Linkatto5903reactions(eachupto200ug)AZK0034CAtto590荧光染料标记试剂盒Lightning-Linkatto5901reaction(upto2mg)AZK0035AAtto594荧光染料标记试剂盒Lightning-Linkatto5943reactions(eachupto20ug)AZK0035BAtto594荧光染料标记试剂盒Lightning-Linkatto5943reactions(eachupto200ug)AZK0035CAtto594荧光染料标记试剂盒Lightning-Linkatto5941reaction(upto2mg)AZK0036AAtto633荧光染料标记试剂盒Lightning-Linkatto6333reactions(eachupto20ug)AZK0036BAtto633荧光染料标记试剂盒Lightning-Linkatto6333reactions(eachupto200ug)AZK0036CAtto633荧光染料标记试剂盒Lightning-Linkatto6331reaction(upto2mg)AZK0037AAtto637荧光染料标记试剂盒Lightning-Linkatto6373reactions(eachupto200ug)AZK0037BAtto637荧光染料标记试剂盒Lightning-Linkatto6371reaction(upto2)mgAZK0038AAtto655荧光染料标记试剂盒Lightning-Linkatto6553reactions(eachupto20ug)AZK0038BAtto655荧光染料标记试剂盒Lightning-Linkatto6553reactions(eachupto200ug)AZK0038CAtto655荧光染料标记试剂盒Lightning-Linkatto6551reaction(upto2mg)AZK0039AAtto680荧光染料标记试剂盒Lightning-Linkatto6803reactions(eachupto20ug)AZK0039BAtto680荧光染料标记试剂盒Lightning-Linkatto6803reactions(eachupto200ug)AZK0039CAtto680荧光染料标记试剂盒Lightning-Linkatto6801reaction(upto2mg)AZK0040AAtto700荧光染料标记试剂盒Lightning-Linkatto7003reactions(eachupto20ug)AZK0040BAtto700荧光染料标记试剂盒Lightning-Linkatto7003reactions(eachupto200ug)AZK0040CAtto700荧光染料标记试剂盒Lightning-Linkatto7001reaction(upto2mg)AZK0041AFluoProbesDyes547H荧光探针染料标记试剂盒Lightning-LinkFluoProbes547H3reactions(eachupto20ug)AZK0041BFluoProbesDyes547H荧光探针染料标记试剂盒Lightning-LinkFluoProbes547H3reactions(eachupto200ug)AZK0041CFluoProbesDyes547H荧光探针染料标记试剂盒Lightning-LinkFluoProbes547H1reaction(upto2mg)AZK0042AFluoProbesDyes594荧光探针染料标记试剂盒Lightning-LinkFluoProbes5943reactions(eachupto20ug)AZK0042BFluoProbesDyes594荧光探针染料标记试剂盒Lightning-LinkFluoProbes5943reactions(eachupto200ug)AZK0042CFluoProbesDyes594荧光探针染料标记试剂盒Lightning-LinkFluoProbes5941reaction(upto2mg)AZK0043AFluoProbesDyes647H荧光探针染料标记试剂盒Lightning-LinkFluoProbes647H3reactions(eachupto20ug)AZK0043BFluoProbesDyes647H荧光探针染料标记试剂盒Lightning-LinkFluoProbes647H3reactions(eachupto200ug)AZK0043CFluoProbesDyes647H荧光探针染料标记试剂盒Lightning-LinkFluoProbes647H1reaction(upto2mg)AZK0044AFluoProbesDyes682荧光探针染料标记试剂盒Lightning-LinkFluoProbes6823reactions(eachupto20ug)AZK0044BFluoProbesDyes682荧光探针染料标记试剂盒Lightning-LinkFluoProbes6823reactions(eachupto200ug)AZK0044CFluoProbesDyes682荧光探针染料标记试剂盒Lightning-LinkFluoProbes6821reaction(upto2mg)AZK0045AFluoProbesDyes752荧光探针染料标记试剂盒Lightning-LinkFluoProbes7523reactions(eachupto20ug)AZK0045BFluoProbesDyes752荧光探针染料标记试剂盒Lightning-LinkFluoProbes7523reactions(eachupto200ug)AZK0045CFluoProbesDyes752荧光探针染料标记试剂盒Lightning-LinkFluoProbes7521reaction(upto2mg)AZK0046APE/Cy5串联染料Lightning-LinkPE/Cy53x10ugAZK0046BPE/Cy5串联染料Lightning-LinkPE/Cy51x100ugAZK0046CPE/Cy5串联染料Lightning-LinkPE/Cy53x100ugAZK0046DPE/Cy5串联染料Lightning-LinkPE/Cy51x1mgAZK0047APE/Cy5.5串联染料Lightning-LinkPE/Cy5.53x10ugAZK0047BPE/Cy5.5串联染料Lightning-LinkPE/Cy5.51x100ugAZK0047CPE/Cy5.5串联染料Lightning-LinkPE/Cy5.53x100ugAZK0047DPE/Cy5.5串联染料Lightning-LinkPE/Cy5.51x1mgAZK0048APE/Cy7串联染料Lightning-LinkPE/Cy73x10ugAZK0048BPE/Cy7串联染料Lightning-LinkPE/Cy71x100ugAZK0048CPE/Cy7串联染料Lightning-LinkPE/Cy73x100ugAZK0048DPE/Cy7串联染料Lightning-LinkPE/Cy71x1mgAZK0049APE/Cy5.5串联染料Lightning-LinkPerCP/Cy5.53x10ugAZK0049BPE/Cy5.5串联染料Lightning-LinkPerCP/Cy5.51x100ugAZK0050APE/TexasRed串联染料Lightning-LinkPE/TexasRed3x10ugAZK0050BPE/TexasRed串联染料Lightning-LinkPE/TexasRed1x100ugAZK0050CPE/TexasRed串联染料Lightning-LinkPE/TexasRed3x100ugAZK0050DPE/TexasRed串联染料Lightning-LinkPE/TexasRed1x1mgAZK0051APE/Atto594串联染料Lightning-Link?PE/Atto5943x10ugAZK0051BPE/Atto594串联染料Lightning-Link?PE/Atto5941x100ugAZK0051CPE/Atto594串联染料Lightning-Link?PE/Atto5943x100ugAZK0051DPE/Atto594串联染料Lightning-Link?PE/Atto5941x1mgAZK0052APerCP/Cy5.5串联染料Lightning-LinkPerCP/Cy5.53x100ugAZK0052BPerCP/Cy5.5串联染料Lightning-LinkPerCP/Cy5.51x1mgAZK0052CPerCP/Cy5.5串联染料Lightning-LinkAPC/Cy5.53x10ugAZK0052DPerCP/Cy5.5串联染料Lightning-LinkAPC/Cy5.51x100ugAZK0052EPerCP/Cy5.5串联染料Lightning-LinkAPC/Cy5.53x100ugAZK0052FPerCP/Cy5.5串联染料Lightning-LinkAPC/Cy5.51x1mgAZK0053ALightning-LinkAPC/Cy7串联染料Lightning-LinkAPC/Cy73x10ugAZK0053BLightning-LinkAPC/Cy7串联染料Lightning-LinkAPC/Cy71x100ugAZK0053CLightning-LinkAPC/Cy7串联染料Lightning-LinkAPC/Cy73x100ugAZK0053DLightning-LinkAPC/Cy7串联染料Lightning-LinkAPC/Cy71x1mgAZK0054A生物素化结合试剂盒typeALightning-LinkBiotinconjugationkittypeA3reactions(upto20μgAb)AZK0054B生物素化结合试剂盒typeALightning-LinkBiotinconjugationkittypeA3reactions(upto200μgAb)AZK0054C生物素化结合试剂盒typeALightning-LinkBiotinconjugationkittypeA1reaction(upto2mgAb)AZK0055A生物素化结合试剂盒typeBLightning-LinkBiotinconjugationkittypeB3reactions(upto20μgAb)AZK0055B生物素化结合试剂盒typeBLightning-LinkBiotinconjugationkittypeB3reactions(upto200μgAb)AZK0055C生物素化结合试剂盒typeBLightning-LinkBiotinconjugationkittypeB1reaction(upto2mgAb)AZK0056A链亲和素标记试剂盒Lightning-LinkStreptavidinconjugationkit3x10ugStreptavidinAZK0056B链亲和素标记试剂盒Lightning-LinkStreptavidinconjugationkit3x100ugStreptavidinAZK0056C链亲和素标记试剂盒Lightning-LinkStreptavidinconjugationkit1x1mgStreptavidinAZK0057A亲和素标记试剂盒Lightning-LinkAvidinconjugationkit3x10ugAvidinAZK0057B亲和素标记试剂盒Lightning-LinkAvidinconjugationkit3x100ugAvidinAZK0057C亲和素标记试剂盒Lightning-LinkAvidinconjugationkit1x1mgAvidinAZK0058A快速荧光素标记Lightning-Link?RapidFluorescein3xAblabelings(each10-20ugscale)AZK0058B快速荧光素标记Lightning-Link?RapidFluorescein3xAblabelings(each100-200ugscale)AZK0058C快速荧光素标记Lightning-Link?RapidFluorescein1xAblabeling(1scale)AZK0059A快速Cy3标记Lightning-Link?RapidCy33xAblabelings(each10-20ugscale)AZK0059B快速Cy3标记Lightning-Link?RapidCy33xAblabelings(each100-200ugscale)AZK0059C快速Cy3标记Lightning-Link?RapidCy31xAblabeling(1scale)AZK0060A快速Cy5标记Lightning-Link?RapidCy53xAblabelings(each10-20ugscale)AZK0060B快速Cy5标记Lightning-Link?RapidCy53xAblabelings(each100-200ugscale)AZK0060C快速Cy5标记Lightning-Link?RapidCy51xAblabeling(1scale)AZK0061A快速Atto488标记Lightning-Link?RapidAtto4883xAblabelings(each10-20ugscale)AZK0061B快速Atto488标记Lightning-Link?RapidAtto4883xAblabelings(each100-200ugscale)AZK0061C快速Atto488标记Lightning-Link?RapidAtto4881xAblabeling(1scale)AZK0062A快速Atto633标记Lightning-Link?RapidAtto6333xAblabelings(each10-20ugscale)AZK0062B快速Atto633标记Lightning-Link?RapidAtto6333xAblabelings(each100-200ugscale)AZK0062C快速Atto633标记Lightning-Link?RapidAtto6331xAblabeling(1scale)AZK0063A快速Atto565标记Lightning-Link?RapidAtto5653xAblabelings(each10-20ugscale)AZK0063B快速Atto565标记Lightning-Link?RapidAtto5653xAblabelings(each100-200ugscale)AZK0063C快速Atto565标记Lightning-Link?RapidAtto5651xAblabeling(1scale)AZK0064A快速Atto594标记Lightning-Link?RapidAtto5943xAblabelings(each10-20ugscale)AZK0064B快速Atto594标记Lightning-Link?RapidAtto5943xAblabelings(each100-200ugscale)AZK0064C快速Atto594标记Lightning-Link?RapidAtto5941xAblabeling(1scale)AZK0065A快速罗丹明标记Lightning-Link?RapidRhodamine3xAblabelings(each10-20ugscale)AZK0065B快速罗丹明标记Lightning-Link?RapidRhodamine3xAblabelings(each100-200ugscale)AZK0065C快速罗丹明标记Lightning-Link?RapidRhodamine1xAblabeling(1scale)AZK0066A快速Atto390标记Lightning-Link?RapidAtto3903xAblabelings(each10-20ugscale)AZK0066B快速Atto390标记Lightning-Link?RapidAtto3903xAblabelings(each100-200ugscale)AZK0066C快速Atto390标记Lightning-Link?RapidAtto3901xAblabeling(1scale)AZK0067A快速DyLight350标记Lightning-Link?RapidDyLight3503xAblabelings(each10-20ugscale)AZK0068A快速DyLight350素标记Lightning-Link?RapidDyLight3503xAblabelings(each100-200ugscale)AZK0069A快速DyLight350标记Lightning-Link?RapidDyLight3501xAblabeling(1scale)AZK0070A快速DyLight405标记Lightning-Link?RapidDyLight4053xAblabelings(each10-20ugscale)AZK0070B快速DyLight405标记Lightning-Link?RapidDyLight4053xAblabelings(each100-200ugscale)AZK0070C快速DyLight405标记Lightning-Link?RapidDyLight405
PerkinElmer提供完整的解决方案包括:试剂、仪器、平台、软件、随时可用的方法、经过全面测试和优化的消耗品以及关于应用的定制式培训。
仪器:元素分析、分子光谱仪、热分析、色谱仪、联用系统、质谱仪
消耗品和附件:元素分析、原子吸收、气象色谱仪、液相色谱、热分析、红外光谱仪、荧光光谱
软件:实验室信息管理系统
2.生物研发(Bio Discovery)
仪器:液体闪烁计数仪、自动化液体处理、微孔板读数仪
试剂:临床诊断试剂、儿童健康试剂、基于细胞的检测试剂、孕妇胎儿健康试剂、放射性化学试剂与放射性治疗、新生儿筛查试剂
消耗品和附件:活细胞成像、液体处理
3.测试和诊断服务(Diagnostics)
特殊诊断:分子测试,孕妇标记物测试,新生儿测试
基因筛查:全自动分辨荧光免疫分析系统,半自动时间分辨免疫分析仪,随机式全自动时间分辨荧光免疫分析系统,串联质谱新生儿筛查仪
PerkinElmer的产品(仪器、检测设备和软件)可以尽早检测出孕期异常。PerkinElmer是唯一一家使用游离 Beta 绒毛膜促性腺激素检测唐氏综合症和染色体 18/13 缺陷的供应商。此项专利技术能够使检出率达到目前该领域的最高水平,让更多家庭提早知情,有备而战。
PerkinElmer提供的新生儿测试试剂种类最为齐全,全新的自动化平台可以同时对几滴血运行多项测试。PerkinElmer一直致力于开发更多的新产品,为母婴健康保驾护航,例如用于检测染色体异常的 BACs on Beads(TM),以及用于评估早产、先兆子痫和胎儿宫内生长迟缓风险的检测试剂盒。
4.医学影像(Medical Imaging)
X射线平板检测仪器:PerkinElmer在非晶硅(a-Si)平板检测器的设计、开发以及制造领域都处于世界的领先地位。所开发的产品广泛应用于人类医学、兽医学、工业无损探伤(NDT)等领域。XRD系列检测器拥有极高的图像分辨率,高达100帧/秒的采集频率,更可以适应20keV~15MeV的高能射线,信息存储读取方便。
PerkinElmer提供XRD系列的2种尺寸平板检测器 - 8英寸(21厘米)和16英寸(41厘米)。每种都有多种选择,如采集速度、能量等级、闪烁体、滤镜、以及采集频率选择等。PerkinElmer也会根据客户的需要提供最适合的型号。
TUNEL的缺点是:1),操作要求高,组织样本需要固定(即使是培养细胞,也需要固定),不恰当的固定方法对实验结果影响很大,导致背景过高或者信号过弱,因此实验结果重复性不好;有的人花上半年做一个体内的TUNEL是一点都不奇怪的。2),大部分诱导凋亡的药物也引起DNA损伤,从而产生DNA断裂,易引入假阳性;3)凋亡晚期细胞基因组大量降解,导致TUNEL标记反而减少,因此此法反应的是早期凋亡比例,不能严格反应凋亡比例,属于半定量研究。
尽管有如此多缺点,但由于其是目前为数不多的能原位标记凋亡细胞的方法,因此用于组织体内凋亡研究仍然是首选。但体外细胞实验研究,很少用此法。
凋亡检测中,TUNEL并不是过时的方法,现在研究凋亡的文献仍然常见。而且相反,还比以前多一点,因为现在体内实验越来越多了,甚至线虫的凋亡研究,都用TUNEL。
凋亡研究中,的确有几种方法过时了,当然是因为有替代方案了,比如DNAladder,电镜。至于AnnexinV是不能用来和TUNEL一起评论的,前者用于细胞,而且主要是悬浮细胞,后者主要用于组织。虽然有人也做镜下的AnnexinV观察,TUNEL的细胞staining,但这都不是主流。
回到lz的原帖,的确如大家所言,做贴壁细胞,不推荐用TUNEL。如果是经典凋亡途径,只是确定比例,用最经典,最常用的subG1法即可,如果是不确定是否是凋亡,用AnnexinV,不过贴壁细胞用此法,要注意消化时间。
其检测原理为:在正常的活细胞中,磷脂酰丝氨酸(phosphotidylserine,PS)位于细胞膜的内侧,但在早期凋亡的细胞中,PS 从细胞膜的内侧翻转到细胞膜的表面,暴露在细胞外环境中。Annexin-Ⅴ(膜联蛋白-V)是一种分子量为35-36KD的Ca2+ 依赖性磷脂结合蛋白,能与PS高亲和力结合。可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜结合。
操作步骤:
1.从室温平衡20min后的铝箔袋中取出所需板条,剩余板条用自封袋密封放回4℃。
2.设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50μL;
3.样本孔中加入待测样本50μL;空白孔不加。
4.除空白孔外,标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μL,用封板膜封住反应孔,37℃水浴锅或恒温箱温育60min。
5.弃去液体,吸水纸上拍干,每孔加满洗涤液(350μL),静置1min,甩去洗涤液,吸水纸上拍干,如此重复洗板5次(也可用洗板机洗板)。
6.每孔加入底物A、B各50μL,37℃避光孵育15min。
7.每孔加入终止液50μL,15min内,在450nm波长处测定各孔的OD值。