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Kerafast Anti-Puromycin抗嘌呤霉素抗体

发布时间:2022-06-11   点击次数:382次

背景介绍:

货号:EQ0001,嘌呤霉素单克隆抗体提供了一种非放射性方法来测量与嘌呤霉素孵育的细胞或组织切片中的整体蛋白质合成(mRNA 翻译)速率,或体内用嘌呤霉素处理的动物。

特色:

* 允许直接使用标准免疫化学方法对翻译进行简单的评估和量化

* 传统脉冲追踪方法的有利替代方法,后者依赖于放射性氨基酸标记

* 与蛋白质印迹和 ELISA 应用兼容

* 使用 Absolute Antibody 的重组平台制造,具有来自杂交瘤 3RH11 的可变区(即特异性)


嘌呤霉素是一种氨基核苷类抗生素,来源于白化链霉菌,在核糖体中发生翻译过程中导致链过早终止。该分子的一部分类似于氨酰化 tRNA 的 3' 端,使其可用于蛋白质翻译分析。用于监测蛋白质合成的经典脉冲追踪或淹没剂量方法依赖于放射性蛋氨酸和()氨酸标记的测量。使用嘌呤霉素免疫检测进行分析是放射性氨基酸标记的一种有利替代方法,并且允许使用标准免疫化学方法直接评估/量化翻译


EQ0001,This monoclonal antibody to puromycin provides a non-radioactive method to measure rates of global protein synthesis (mRNA translation) in cells or tissue slices incubated with puromycin, or animals treated with puromycin in vivo.

Highlights:

* Allows for the simple evaluation and quantification of translation directly using standard immunochemical methods

* Advantageous alternative to traditional pulse-chase methods, which rely on radioactive amino acid labeling

* Compatible with Western Blot and ELISA applications

* Manufactured using Absolute Antibody’s Recombinant Platform with variable regions (i.e., specificity) from the hybridoma 3RH11

Puromycin is an aminonucleoside antibiotic, derived from the Streptomyces alboniger bacterium, that causes premature chain termination during translation taking place in the ribosome. Part of the molecule resembles the 3' end of the aminoacylated tRNA, making it useful for protein translation analysis. Classical pulse-chase or flooding dose methods used to monitor protein synthesis rely on the measurement of radioactive methionine and cysteine labels. Analysis using puromycin immunodetection is an advantageous alternative to radioactive amino acid labeling, and allows for the evaluation/quantification of translation directly using standard immunochemical methods


Product Type:Antibody
Name:Anti-Puromycin (3RH11)
Host:Mouse
Isotype:IgG1 kappa
Clonality:Monoclonal
Clone Name:3RH11
Specificity:This antibody recognizes puromycin.
Immunogen:puromycin hydrochloride
Format:Liquid
Purification Method:Protein G purified
Buffer:PBS with 0.02% Proclin 300
Tested Applications:Western blotting (1:1,000), ELISA and Immunofluorescence microscopy.
Storage:+4C (short-term), -20C (long-term); Avoid repeated freeze/thaw cycles.
Shipped:Cold packs


数据展示:


(A) C2C12 myoblasts were starved of serum and leucine for 2 hr and then IGF-1 and leucine were added to the medium of some of the cells for 45 min. Puromycin (1uM) was added to the medium of some of the cells (lanes 3-6) 30 min before harvest. (B) Quantification of western blot analysis from panel A. (C) In the same study, but in a separate set of culture dishes, cells were incubated with [35S]methionine instead of puromycin and incorporation was measured.



A Western blot was run using the same samples where one set was run on the left side of the gel and the other on the right.  The left side was probed with our original monoclonal anti-puromycin antibody and the other side was probed with the recombinant anti-puromycin antibody, both at 1:1,000 dilution. The secondary antibody was used at the same dilution for both sides and they were both exposed for ~40 sec. The first two samples were from skeletal muscle of mice where the first lane is muscle from the control hindlimb and the other is from a hindlimb that had been immobilized with a cast for three days.  The other 3 lanes are from HEK393T cells: the first lane is from cells incubated in complete medium, the middle lane is from cells incubated for 2 hr in medium lacking glucose and serum, and the last lane is cells incubated for 1.5 hr without glucose or serum and then glucose and serum were returned during the last 30 min.  As expected, puromycin incorporation was lower in the immobilized hindlimb compared to the contralateral control hindlimb, and also in cells deprived of glucose and serum compared to cells in complete medium.  Resupplementation partially restored incorporation.



参考文献:

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2. Tom Dieck S, Kochen L, Hanus C, Heumüller M, Bartnik I, Nassim-Assir B, Merk K, Mosler T, Garg S, Bunse S, Tirrell DA, Schuman EM. Direct visualization of newly synthesized target proteins in situ. Nat Methods. 2015 May;12(5):411-4.

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4. Rangaraju V, Lauterbach M, Schuman EM. Spatially Stable Mitochondrial Compartments Fuel Local Translation during Plasticity. Cell. 2019 Jan 10;176(1-2):73-84.e15.

5. Hafner AS, Donlin-Asp PG, Leitch B, Herzog E, Schuman EM. Local protein synthesis is a ubiquitous feature of neuronal pre- and postsynaptic compartments. Science. 2019 May 17;364(6441).

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