Name: multiplexed single-molecule force proteolysis measurements using magnetic tweezers
Uploaded: 2012-07-25T11:00:00
Description: Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

Bu çalışma Bilimsel Arabirimi (ARD), NIH Direktörü Yeni Yenilikçisi Ödülü Programı çerçevesinde Ulusal Sağlık Enstitüleri 1-DP2-OD007078 (ARD), William Bowes Jr Stanford Yüksek Lisans Bursu (ASA de Burroughs Wellcome Kariyer Ödülü ile desteklenmiştir ) ve Stanford Kardiyovasküler Enstitüsü Younger predoctoral Bursu (JC). Yazarlar mikroskopi ekipman loaning James Spudich teşekkür ederim.

1. Akış Hücre Hazırlık <ol><li> Lamelleri (# 1.5, 22x22 mm ve 22x40 mm, VWR) sonication kullanılarak temizlenir. <ol><li> Lamelleri tutarak ve (bkz: adım 2) sonikatör uyum gösterme yeteneğine sahip küçük bir cam kaba lamelleri ekleyin. </li><li> Izopropanol ile kabın doldurmak ve 20 dakika boyunca bir banyo içinde sonikatör ses dalgalarına maruz. </li><li> Izopropanol atın ve bir Barnsted MilliQ aparatı veya benzer bir cihaz tarafından üretilen deiyonize bol su ile lamelleri durulayın. Su kabı d...

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Bu protokol, klasik bir tek bir molekül tekniği için yeni bir kullanımını açıklar. Manyetik cımbız, bir maliyet-etkin şekilde yüksek verimli tek bir molekül testleri orta sağlar. Ancak, tüm deneysel teknikler gibi zorluklar ve potansiyel tuzaklar vardır. Manyetik cımbız Sınırlamalar Optik bir tuzak ile karşılaştırıldığında MT aygıtının mekansal ve zamansal çözünürlüğü düşük. Bundan başka, burada açıklanan basit MT tarafın...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> Mikro Kapak Cam # 1.5 (22x22) </td><td> VWR </td><td> 48366-067 </td></tr><tr><td> Mikro Kapak Cam # 1.5 (22x40) </td><td> VWR </td><td> 48393-048 </td></tr><tr><td> Lambda DNA </td><td> Invitrogen </td><td> 25250-010 </td></tr><tr><td> T4 DNA ligaz </td><td> Invitrogen </td><td> 15224-041 </td></tr><tr><td> Microcon Ultracel YM-100 </td><td> Millipore </td><td> 42413 </td></tr><tr><td> Anti-digoxigenin </td><td> Roche Diagnostics </td><td> 11-333-089-001 </td></tr><tr><td> Tween 20 </td><td> Sigma </td><td> P9416-100ML </td></tr&gt; <tr><td> Anti-myc Antikor </td><td> Invitrogen </td><td> 46-0603 </td></tr><tr><td> Bovine Serum Albumin </td><td> Sigma </td><td> B4287-5G </td></tr><tr><td> Dynabeads M-280 Streptavidin </td><td> Invitrogen </td><td> 658.01D </td></tr><tr><td> Dynabeads MyOne T1 Streptavidin </td><td> Invitrogen </td><td> 658.01D </td></tr><tr><td> p-Aminophenylmercuric asetat </td><td> Calbiochem </td><td> 164610 </td></tr><tr><td> Biotin-maleimid </td><td> Sigma Aldrich </td><td> B1267 </td></tr><tr><td> Biotin oligo etiketli </td><td> IDT DNA </td><td> Özel sentezi </td></tr><tr><td> Digoxigenin oligo etiketli </td><td> IDT DNA </td><td> Özel sentezi </td></tr><tr><td> Kollajenpeptid gen </td><td> DNA 2.0 </td><td> Özel sentezi </td></tr><tr><td> MMP-1 cDNA </td><td> Harvard Plazmid Veritabanı </td><td></td></tr><tr><td> z-çevirmen </td><td> Thorlabs </td><td> MTS50 </td></tr><tr><td> Çevirmen için servo kontrol </td><td> Thorlabs </td><td> TDC001 </td></tr></tbody></table>

multiplexed single-molecule force proteolysis measurements using magnetic tweezers

Mekanik güçlerin üretme ve algılama kanser metastazı 1, aterogenez 2 ve 3 yara iyileşmesi ile doğrudan ilgili olan, hücre fizyolojisi her yerde görülen bir yönüdür. Bu örneklerin her birinde, hücreler çevreleri üzerinde kuvvet uygularlar ve aynı enzimatik remodel ekstraselüler matriks (ECM) hem de. ECM kuvvetlerin etkisi böylece 4-7 büyük olasılıkla onun biyolojik ve tıbbi önemi nedeniyle önemli bir ilgi alanı haline gelmiştir. Gibi optik tuzaklama 8, atomik kuvvet mikroskobu 9, ve manyetik cımbız 10,11 olarak tek bir molekülün teknikleri araştırmacılar bireysel proteinler üzerinde kuvvetleri uygulayarak moleküler düzeyde enzim fonksiyonu soruşturma için izin. Bu tekniklerin, manyetik cımbız (MT) Düşük maliyet ve yüksek verimlilik için dikkat çekicidir. MT ~ 1-100 pN aralığında kuvvetleri uygulamak ve milisaniye zamansal çözünürlük sağlayabilir,ayrıca tek moleküllü seviyesi 12 az enzim mekanizmasının çalışma eşleştirilir nitelikleri. Burada tek bir protein moleküllerinin proteoliz tarihinde yürürlüğe etkisini incelemek için son derece parallelizable MT tahlil raporu. Bu matris metalloproteinaz 1 (MMP-1) tarafından bir trimeric kolajen peptid proteolizin spesifik bir örnek sunmak; Bununla birlikte, bu tahlilde kolayca diğer substratlar ve proteazlar incelemek için adapte edilebilir.

Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

Bu yazıda son derece parallelizable şekilde tek bir molekül düzeyinde enzimatik proteolizin yürürlüğe etkisini incelemek için manyetik cımbız kullanımı açıklanır.

Manyetik cımbız kullanarak Multiplexed Tek molekül Kuvvetleri Proteoliz Ölçümleri

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Research

JoVE Journal

Bioengineering

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Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

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Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

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Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Magnetic Tweezers for the Measurement of Twist and Torque

Büküm ve Moment Ölçümü için Manyetik Cımbız

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

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Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Planar on Tek Molekül Floresans Mikroskopi bilayers Desteklenen

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

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Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

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We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

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Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

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Yüksek Konsantrasyonlı Tek Moleküllü Mikroskopi için Sıfır Mod Dalga Kılavuzlarının İmalatı

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...