Kerafast抗體Anti-Puromycin [3RH11]

背景介紹：

貨號：EQ0001，嘌呤霉素單克隆抗體提供了一種非放射性方法來測量與嘌呤霉素孵育的細(xì)胞或組織切片中的整體蛋白質(zhì)合成（mRNA 翻譯）速率，或體內(nèi)用嘌呤霉素處理的動物。

特色：

* 允許直接使用標(biāo)準(zhǔn)免疫化學(xué)方法對翻譯進(jìn)行簡單的評估和量化

* 傳統(tǒng)脈沖追蹤方法的有利替代方法，后者依賴于放射性氨基酸標(biāo)記

* 與蛋白質(zhì)印跡和 ELISA 應(yīng)用兼容

* 使用 Absolute Antibody 的重組平臺制造，具有來自雜交瘤 3RH11 的可變區(qū)（即特異性）

嘌呤霉素是一種氨基核苷類抗生素，來源于白化鏈霉菌，在核糖體中發(fā)生翻譯過程中導(dǎo)致鏈過早終止。該分子的一部分類似于氨酰化 tRNA 的 3' 端，使其可用于蛋白質(zhì)翻譯分析。用于監(jiān)測蛋白質(zhì)合成的經(jīng)典脈沖追蹤或淹沒劑量方法依賴于放射性蛋氨酸和（半）氨酸標(biāo)記的測量。使用嘌呤霉素免疫檢測進(jìn)行分析是放射性氨基酸標(biāo)記的一種有利替代方法，并且允許使用標(biāo)準(zhǔn)免疫化學(xué)方法直接評估/量化翻譯

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

 

數(shù)據(jù)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.

Kerafast抗體Anti-Puromycin [3RH11]已發(fā)表文獻(xiàn)

參考文獻(xiàn)：

1. Lee KH, Zhang P, Kim HJ, Mitrea DM, Sarkar M, Freibaum BD, Cika J, Coughlin M, Messing J, Molliex A, Maxwell BA, Kim NC, Temirov J, Moore J, Kolaitis RM, Shaw TI, Bai B, Peng J, Kriwacki RW, Taylor JP. C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles. Cell. 2016 Oct 20;167(3):774-788.e17.

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.

3. Gantois I, Khoutorsky A, Popic J, Aguilar-Valles A, Freemantle E, Cao R, Sharma V, Pooters T, Nagpal A, Skalecka A, Truong VT, Wiebe S, Groves IA, Jafarnejad SM, Chapat C, McCullagh EA, Gamache K, Nader K, Lacaille JC, Gkogkas CG, Sonenberg N. Metformin ameliorates core deficits in a mouse model of fragile X syndrome. Nat Med. 2017 Jun;23(6):674-677.

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).

6. Hörnberg H, Pérez-Garci E, Schreiner D, et al. Rescue of oxytocin response and social behaviour in a mouse model of autism. Nature. 2020;584(7820):252-256.

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