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产品信息
|
产品名称 |
产品编号 |
规格 |
价格 |
|
FITC Phalloidin FITC标记鬼笔环肽 |
40735ES75 |
300 T |
1533.00 |
背景描述
鬼笔环肽（Phalloidin）是一种来源于毒蕈类鬼笔鹅膏（Amanita phalloides）的环状七肽毒素，以高亲和力（Kd= 20 nM）选择性结合于丝状肌动蛋白F-actin，而不会与单体肌动蛋白G-actin结合，通常用来标记组织切片，细胞培养物或无细胞体系中的F-actin，从而对F-actin进行定性和定量分析。另外，鬼笔环肽衍生物也以相近的亲和力结合于大小纤维，无论是动植物来源的肌肉细胞或非肌肉细胞，按照每一个肌动蛋白亚基约与一个鬼笔环肽分子的计量比结合。非特异性结合几乎可忽略，染色区域和非染色区域辨识度非常明显。因此，鬼笔环肽衍生物特别适合替代肌动蛋白（Actin）抗体进行相关研究。另外鬼笔环肽衍生物很小，直径约12-15Å，分子量＜2000 Daltons，未标记肌动蛋白（Actin）的许多生理特性都得以维持，比如，同肌动蛋白结合蛋白如肌球蛋白，原肌球蛋白，DNase I等仍能发生反应；鬼笔环肽标记的纤维丝仍可穿透固相肌球蛋白基质；以及甘油抽提的肌纤维标记后仍可收缩等。
鬼笔环肽（Phalloidin）的结合阻止丝状肌动蛋白（微丝）的解离，稳定微丝结构，从而破坏微丝的聚合-去聚合的动态平衡。此特性使得肌动蛋白聚合发生的临界浓度（CC）降至＜1 µg/mL，因此，可用作一种聚合促进剂。此外，鬼笔环肽还可抑制F-actin的ATP水解活性。
本品为FITC标记的鬼笔环肽，染色反应特异性强，对比性高，具有比Actin抗体更好的染色效果，适合用作F-actin的定性和定量检测。另外，经本品结合后的F-actin仍能维持actin自身具有的许多生物学特性。且本品的结合没有物种差异性，适用性广泛。
我司提供储存液形式（浓度为20 µM）的FITC标记鬼笔环肽，用户根据自身需求选择，建议使用浓度为80~200 nM。
产品性质
|
分子式（Molecular Formula） |
C56H60N10O15S2 |
|
分子量（Molecular Weight） |
1177.3 |
|
最大激发/发射波长（Ex/Em） |
495~496/513~516 nm |
|
多肽序列（Sequence） |
FITC-bicyclic(Ala-DThr-Cys-cis-4-hydroxy-Pro-Ala-2-mercapto-Trp-4-hydroxy-5-amino-Leu)(S-3 to 6) |
|
外观（Appearance） |
淡黄色至黄色溶液 |
运输与保存方法
冰袋运输。-20 ℃避光干燥保存，1年有效。
注意事项
1）鬼笔环肽具有毒性，需小心操作（对人的半数致死剂量LD50约2 mg/kg）。
2）为了您的安全和健康，请穿实验服并戴一次性手套操作。
3）本产品仅作科研用途！
需要自备材料
1）（可选）甲醇
2）1×PBS缓冲液，pH 7.4，细胞培养级别
3）固定液4%多聚甲醛（溶于PBS缓冲液）
4）丙酮或透化液0.5% Triton X-100（溶于PBS缓冲液）
5）Fluoromount-GTM 水溶性封片剂（不含DAPI）（货号：36307ES08），DAPI（货号：40727ES10）
6）（可选）DAPI Fluoromount-GTM 水溶性封片剂（含DAPI）（货号：36308ES11）
7）（可选）BSA，标准级别（货号：36101ES25）
8）载玻片和盖玻片
9）盖玻片周围密封液（如透明指甲油）
10）组装有FITC激发/发射滤片，以及DAPI激发/发射滤片的荧光显微镜或共聚焦显微镜
操作步骤
1. 工作液准备
本品以溶于甲醇的20 µM储存液形式提供，总量为300 µL。按照100 nM的工作液浓度来换算，可制备总量为60 mL的工作液。建议收到产品后，根据单次使用量，对母液进行小量分装，-20 ℃避光冻存，一年稳定。
开始实验前，使用1×PBS缓冲液稀释储存液到需要的工作浓度。推荐工作浓度为：80~200 nM。工作液现配现用。
2. 染色步骤
1）细胞爬片生长24 h，使其密度达到50%汇合度。
2）吸掉培养液，37℃预热的1×PBS（pH 7.4）清洗细胞2次。
3）使用溶于PBS的4%甲醛溶液进行细胞固定，室温固定10 min。
注意：避免固定剂中含有甲醇成分，因为甲醇在固定过程中可能破坏肌动蛋白。
4）室温条件下，用PBS清洗细胞2~3次，每次10 min。
5）室温条件下，用丙酮（≤-20℃）脱水或者用0.5% Triton X-100溶液透化处理5 min。
6）室温条件下，用PBS清洗细胞2~3次，每次10 min。
7）取200 µL配制好的 FITC标记鬼笔环肽工作液，覆盖住盖玻片上的细胞，室温避光孵育30 min（通常情况下，4 ℃~37 ℃孵育皆可）。
注意：为了降低背景，可于FITC标记的鬼笔环肽工作液内加入1% BSA；另外，孵育过程中为了避免溶液挥发，可将盖玻片转移到一个密封的容器内。
8）用PBS清洗盖玻片3次，每次5 min。
9）使用200 µL DAPI溶液（浓度：100 nM）对细胞核进行复染，约30 s。
10）用PBS清洗盖玻片，然后倒置在已经滴有一滴Fluoromount-GTM 水溶性封片剂的载玻片上。使用纸巾轻轻檫掉多余封片剂，然后用指甲油永久封片。此法制备的标本玻片可置于4 ℃避光保存，通常6个月内可继续做F-actin染色分析。
注意：也可以直接使用含有DAPI的抗荧光淬灭封片剂（货号：36308ES11）合并步骤9）10），简化步骤。
- 荧光显微镜或者共聚焦显微镜下进行荧光观察，选择FITC激发/发射滤片（Ex/Em=496/516 nm）和DAPI激发/发射滤片（Ex/Em=364/454 nm）。
HB240410
Q：为什么我激发后荧光很快就猝灭了？
A：可能是使用浓度过高造成，可以再适当降低一些试剂浓度；可以在寻找视野时使用低能量的激光，待找到合适视野时使用高能量激光激发观察。2.鬼笔环肽不能和点击反应（例：EDU）共染色，否则鬼笔环肽没荧光信号。
Q：该试剂可以应用于组织切片吗？
A：石蜡切片或者冰冻切片都不建议，可能效果不好，建议使用抗肌动蛋白的抗体。
Q：每次是添加多少试剂量（工作液）进行荧光显微镜检测？
A：建议添加的量是能够完全浸没细胞即可，不同的细胞染色情况不同，相应鬼笔环肽使用量也需根据不同情况而定。
Q：几种不同的鬼笔环肽的区别是什么，如何选择？
A：是荧光基团的不同，激发出的荧光颜色不同，用于区分共染时其他荧光染料的荧光颜色，对于选择需要机器满足激发和发射波长要求。
Q：细胞处理时，须用BSA 封闭吗？
A：是的，这样做可以降低背景色。
Q：鬼笔环肽染色存在种属区分吗？
A：对于对固定的细胞染色，不区分种属。
Q：想染植物细胞微丝，鬼笔环肽能不能使用？
A：由于植物含细胞壁，因此在未破坏细胞壁时染色进入细胞内较为困难，因此对于含细胞壁细胞染色可以去除细胞壁制备原生质体进行染色。
Q：染色后多久进行上机观察检测，可以放置过夜吗？
A：不推荐，染色后应尽量在 60min 内完成上机观察和检测过程。
Q：关于几种产品的状态及含量问题介绍？
A：iFluor 系列的 40737、40762、40736 均为粉末，40734 和 40735 均为液体形式提供。对于 300T
的含量不是指含有 300ul，具体的 300T 是指能检测 300 次左右。
Q：我用鬼笔环肽染小鼠的巨噬细胞，发现鬼笔环肽没有进入巨噬细胞中，而是在巨噬细胞的表面，
有什么更好的建议嘛？
A：巨噬细胞染色效果较差一些，建议延长透化时间，例如用丙酮（≤-20℃）脱水或者用 0.5% Triton X-100 溶液透化处理 10-15min。
Q：鬼笔环肽可以染活细胞，以及染色后可以用流式检测嘛？
A： 不推荐，荧光标记的鬼笔环肽不具有细胞透性，因此没有被广泛用于活细胞标记 。
Q：我的细胞过表达G-actin，会不会影响鬼笔环肽的染色效果？
A：不会影响鬼笔环肽的染色效果。因为鬼笔环肽只选择性结合于丝状肌动蛋白 F-actin，而不会与
单体肌动蛋白 G-actin 结合。
Q：鬼笔环肽可以双标染色吗？
A：可以，例如可以与 DAPI 溶液对细胞核进行复染。
Q：鬼笔环肽可以跟免疫荧光同时做吗？
A：可以和抗体共染，可以先染抗体，再做鬼笔环肽的染色。
Q：可以用那些溶液来固定细胞，要注意什么？
A：可以用溶于PBS 的 4%多聚甲醛溶液进行细胞固定，固定时避免固定剂中含有甲醇成分，因为甲醇
在固定过程中可能破坏肌动蛋白F-actin。
Q：鬼笔环肽染色后，封片保存在 4 度冰箱一段时间后，再检测信号可以吗？
A：不建议这样操作，因为封片后鬼笔环肽的荧光信号会逐渐减弱。建议现加现测。
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