高通量單分子力譜儀系統(tǒng)

德國(guó)AFS單分子原子力譜儀 (Single Molecule Atomic Force Spectrometer)

新型，高線性原子力光譜儀，用于蛋白質(zhì)動(dòng)力學(xué)的定量測(cè)量
該裝置可對(duì)單個(gè)分子進(jìn)行高通量分析，闡明諸如解折疊速率和保持其構(gòu)象的鍵強(qiáng)度等特征。根據(jù)要確定的分子的性質(zhì)，此設(shè)備可以與多種協(xié)議一起使用：力擴(kuò)展，力鉗，力斜坡和各種重新折疊協(xié)議。

• 力鉗和力-延伸
• 亞納米分辨率
• 亞毫秒級(jí)時(shí)間分辨率
• 蛋白質(zhì)折疊和展開
• 鍵裂解和形成
• 全自動(dòng)操作
• 強(qiáng)大的分析軟件
• 簡(jiǎn)單的用戶界面
• 高通量

原子力光譜儀（AFS）是用于在校準(zhǔn)的機(jī)械負(fù)載下單個(gè)蛋白研究的儀器.
AFS用于了解機(jī)械力在整個(gè)生物光譜，影響蛋白質(zhì)的動(dòng)力學(xué)和化學(xué)性質(zhì)。
AFS允許拾取和機(jī)械操縱單個(gè)重組蛋白。 該系統(tǒng)全自動(dòng)，可以連續(xù)運(yùn)行數(shù)天，無需人看管。
操作軟件中包含的例程明確識(shí)別所研究蛋白質(zhì)的機(jī)械指紋，并因此能夠
自動(dòng)識(shí)別和存儲(chǔ)數(shù)據(jù)。在為期一天的實(shí)驗(yàn)結(jié)束后，可以收集多個(gè)一百種單蛋白痕跡。

用于AFS測(cè)量的蛋白質(zhì)附著的標(biāo)準(zhǔn)方法是使用硫醇化學(xué)方法，該方法是通過在覆蓋有金的玻璃蓋玻片上將在末端含有半胱氨酸錨定分子的重組蛋白分層來獲得的。 zui有效的方法是結(jié)合使用HaloTag（Promega）技術(shù)和硫醇化學(xué)方法，該方法可以很容易地將共價(jià)連接的蛋白質(zhì)傳遞給兩者。 鍍金的懸臂和Halo-配體的玻璃蓋玻片。 共價(jià)連接的蛋白質(zhì)可以長(zhǎng)時(shí)間進(jìn)行機(jī)械操作。
優(yōu)選的蛋白質(zhì)樣品被安排為串聯(lián)模塊蛋白質(zhì)。 當(dāng)此類多蛋白通過AFS鋪展時(shí)，其作用力是*的機(jī)械指紋，可將它們與困擾單分子研究的更常見的非特異性事件明確區(qū)分開。

This unique Atomic-Force-Spectroscope (AFS) , based on the Atomic Force Microscope Technology (AFM) was specially designed for studying single proteins placed under a calibrated mechanical load.
The upside down design, where the cantilever, laser and sensor is fixed while the substrate with attached protein is moved by a ultra-precise linear Piezo, enables us to get a feedback response time better than 1ms and a position accuracy better than 1nm.
The software is based on IGOR and enables the operator to run automatic experiments over the day without putting hands on.
The AFS can be operated in two modes:
- force extension (constant velocity)
- force clamp (constant force)
In force clamp mode the operator can design his own force protocol to measure protein folding, unfolding and protein chemical reactions.
The software includes routines that unambiguously identify the mechanical fingerprints of the protein being studied, and thus is able to recognize and store data automatically.
A tuneable Proportional-Integral-Differential system (PID) enables the operator to adjust the parameter for the Piezo, cantilever and probe to get the best feedback time for each individual experiment.
The AFS comes as a complete workstation, incl. vibration damping table, PC Monitor and software.
For further information please visit the web page of Professor Julio Fernandez, in which collaboration this system was designed.

We use a purpose built single-molecule force spectroscope (SMFS) developed by the Fernandez lab at Columbia University and built by Luigs Neumann.[1] This device allows for high throughput analysis of single molecules, elucidating characteristics such as the rate of unfolding and the strength of the bonds holding its conformation. This device can be used with a range of protocols depending on the property of the molecule to be determined: force-extension, force-clamp, force-ramp, and various refolding protocols.

Chimera Fingerprinting

One of the main difficulties with high-throughput SMFS is that a great proportion of the data produced is not relevant for study as it does not the display the molecule under investigation. Non-specific binding of the cantilever tip at various points along the molecule leads to incomplete traces. One method to overcome this is to add specific immunoglobulin (Ig) domains at either end of the molecule being studied; these Ig domains unfold with very specific patterns which, if present in a trace, indicate the molecule has been correctly stretched. Thus thousands of force-extension curves can be sorted rapidly by requiring them to have the characteristic ‘saw-tooth’ pattern that indicative of the unfolding of an Ig domain.

Surface Attachment

In addition to using flanking molecules to improve the detection of correct stretching traces, surface chemistry can be used to covalently bind the end of the protein construct to surface or the tip. This increases the likelihood that a molecule will be stretched along its full length and thus increases the efficiency of the experiment. The most common way to do this is to prepare the surface such that it contains a particular  ligand for an enzyme. This enzyme is then attached at the end of the protein construct, similarly to the fingerprint Ig domains. As such when the protein is deposited on the surface, the enzyme reacts with the functionalized surface to produce a strong covalent attachment between the surface and the protein construct.[2]

[1] Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy

Ionel Popa  Pallav Kosuri  Jorge Alegre-Cebollada  Sergi Garcia-Manyes  Julio M. Fernandez

Nature Protocols June, 2013

[2] Nanomechanics of HaloTag Tethers

Ionel Popa  Ronen Berkovich  Jorge Alegre-Cebollada  Carmen L. Badilla  Jaime Andrés Rivas-Pardo  Yukinori Taniguchi  Masaru Kawakami  Julio M. Fernandez

Journal of the American Chemical Society August, 2013

Animation of AFS unfolding of a talin-Ig construct