生物素标记EMSA探针－HIF-1α

Biotin-labeled EMSA probe-HIF-1α

B752129-200μl

200μL

0.2μM

-20°C储存

超低温冰袋运输

-20℃保存，一年有效。

生物素标记EMSA探针－HIF-1α是用于EMSA(也称gelshift)研究的并经生物素(Biotin)标记的HIF-1αconsensusoligonucleotide。这个生物素标记的双链寡核苷酸含有公认的HIF-1α结合位点，可以用作EMSA研究时的探针。HIF-1α consensus oligo的序列如下：5'-TCT GTA CGT GAC CAC ACT CAC CTC-3'","3'-AGA CAT GCA CTG GTG TGA GTG GAG-5'",'本生物素标记EMSA探针已经过纯化，可以直接用于EMSA结合反应。本生物素标记EMSA探针可以和阿拉丁的化学发光法EMSA试剂盒配套使用。一个包装的生物素标记探针可以进行约200-400个样品的EMSA检测。 注意事项 ： 避免加热到40℃以上，温度过高会导致双链DNA探针解聚成单链。而单链无法用于EMSA研究。对于基于生物素标记的EMSA检测的详细操作可以参考阿拉丁的化学发光法EMSA试剂盒的使用说明。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.本生物素标记EMSA探针用于EMSA结合反应时，参考如下步骤进行：A.如下设置EMSA结合反应：阴性对照反应： Nuclease-Free Water7-7.5μlEMSA/Gel-Shift 结合缓冲液(5X)2μl细胞核蛋白或纯化的转录因子0μl生物素标记探针0.5-1μl总体积10μl样品反应： Nuclease-Free Water5-5.5μlEMSA/Gel-Shift 结合缓冲液(5X)2μl细胞核蛋白或纯化的转录因子2μl生物素标记探针0.5-1μl总体积10μl探针冷竞争反应： Nuclease-Free Water4-4.5μlEMSA/Gel-Shift 结合缓冲液(5X)2μl细胞核蛋白或纯化的转录因子2μl未标记的探针1μl生物素标记探针0.5-1μl总体积10μl突变探针的冷竞争反应： Nuclease-Free Water4-4.5μlEMSA/Gel-Shift 结合缓冲液(5X)2μl细胞核蛋白或纯化的转录因子2μl未标记的突变探针1μl生物素标记探针0.5-1μl总体积10μlSuper-shift反应： Nuclease-Free Water4-4.5μlEMSA/Gel-Shift 结合缓冲液(5X)2μl细胞核蛋白或纯化的转录因子2μl目的蛋白特异抗体1μl生物素标记探针0.5-1μl总体积10μl注：生物素标记EMSA探针的推荐用量为每个反应0.5微升，如果检测出来的目的蛋白的EMSA条带偏弱，可以适当加大生物素标记EMSA探针的用量至0.75微升或1微升。注2：对于冷竞争时使用的未标记的探针或未标记的突变探针，使用量可以根据实际情况调整使用的体积。推荐的用于冷竞争的未标记的探针或突变探针的用量为生物素标记探针的50-100倍。B.按照上述顺序依次加入各种试剂，在加入标记好的探针前先混匀，并且室温(20-25℃)放置10分钟，从而消除可能发生的探针和蛋白的非特异性结合，或者让冷探针优先反应。然后加入标记好的探针，混匀，室温(20-25℃)放置20分钟。C.加入1μl EMSA/Gel-Shift上样缓冲液(无色，10X)，混匀后立即上样。注意：有些时候溴酚蓝会影响蛋白和DNA的结合，建议尽量使用无色的EMSA/Gel-Shift上样缓冲液。如果对于使用无色上样缓冲液在上样时感觉到无法上样，可以在无色上样缓冲液里面添加极少量的蓝色的上样缓冲液，至可以观察到蓝颜色即可。2.对于基于生物素标记的EMSA检测的更多详细操作可以参考阿拉丁的化学发光法EMSA试剂盒的使用说明。 Aladdin's Biotin-labeled EMSA Probe - HIF-1α (0.2µM) is a HIF-1α consensus oligonucleotide designed for EMSA . This double-stranded oligonucleotide contains the well-recognized binding sequence for hypoxia-inducible factor 1-alpha (HIF-1α), and can be used as a probe for EMSA. The nucleotide sequence of HIF-1α consensus oligo is as follows Precautions ： Avoid heating above 40℃, as double-stranded DNA probe will be denatured at high temperatures into single strands that cannot be used in EMSA.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use ： 1.EMSA binding reactiona.Set up the following reactions by adding the reagents in sequence. Negative Control Reactions: Nuclease-Free Water7-7.5μlEMSA/Gel-Shift Binding Buffer (5X)2μlNuclear Protein or Purified Transcription Factor0μlBiotin-labeled Probe0.5-1μlTotal Volume10μlSample Reaction:Nuclease-Free Water5-5.5μlEMSA/Gel-Shift Binding Buffer (5X)2μlNuclear Protein or Purified Transcription Factor2μlBiotin-labeled Probe0.5-1μlTotal Volume 10μlCold Probe Competition Reaction:Nuclease-Free Water4-4.5μlEMSA/Gel-Shift Binding Buffer (5X)2μlNuclear Protein or Purified Transcription Factor2μlUnlabeled Probe1μlBiotin-labeled Probe0.5-1μlTotal Volume 10μlCold Mutant Probe Competition Reaction: Nuclease-Free Water4-4.5μlEMSA/Gel-Shift Binding Buffer (5X)2μlNuclear Protein or Purified Transcription Factor2μlUnlabeled Probe1μlBiotin-labeled Probe0.5-1μlTotal Volume10μlSuper-shift Reaction:Nuclease-Free Water4-4.5μlEMSA/Gel-Shift Binding Buffer (5X)2μlNuclear Protein or Purified Transcription Factor2μlTarget Protein Specific Antibody1μlBiotin-labeled Probe0.5-1μlTotal Volume10μlNote 1: The recommended dosage of biotin-labeled EMSA probe is 0.5µl per reaction. If the EMSA bands of target proteins are weak, the dosage of biotin-labeled EMSA probe can be increased to 0.75 µl or 1 µl as appropriate. Note 2: For cold probe competition, the recommended amount of unlabeled probe is 50-100 times that of the biotin-labeled probe, but can be adjusted as required.b.Before adding the labeled probe, the reaction mix should be mixed well and incubated at room temperature (20-25℃) for 10 minutes to eliminate possible non-specific binding between probes and proteins. Otherwise allow cold probes react preferentially. Then add the labeled probe, mix well and incubate at room temperature (20-25℃) for 20 minutes.c.Add 1μl of EMSA/Gel-Shift Loading Buffer (colorless, 10X) into each reaction and load onto gel immediately after mixing. Note: Sometimes bromophenol blue can affect protein-DNA interactions. It is recommended to use the colorless EMSA/Gel-Shift Loading Buffer, or that including a very small amount of blue loading buffer to make observation easier.2.For more detailed EMSA procedures with the biotin-labeled probes, please refer to the manual of ’s Chemiluminescent EMSA Kit