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X-ray crystallography (以X-ray晶體學測定蛋白質結構)

1. Cloning
2. Protein expression
3. Protein purification
4. Crystallization
5. Data collection
6. Solve phase problem
7. Protein structure

Nucleic acids binding protein-related biochemical studies
(核酸結合蛋白相關之生化與分生實驗)

Clone

利用PCR將目標基因放大，並將insert DNA接入Plasmid DNA之中，即可完成表達載體。Amplified the target gene by PCR technique and insert the DNA into the plasmid to prepare an expression vector with a specific gene.

Nuclease activity assay

以TBE-Urea PAGE或 Agarose gel分析核酸受質被降解之程度，用於判斷蛋白降解核酸的效率。
We measure the nuclease activity by observing the degrading bands of DNA in TBE-Urea PAGE or agarose gel.

DNA binding assay - EMSA

利用Non-denature gel 分析，以不破壞蛋白質與DNA結構的方式觀察兩者之間結合的效率。
We use Electrophoretic Mobility Shift Assay (EMSA) to measure the interaction between a protein and DNA.

Quantitative binding ability analysis between target proteins and nucleic acids/drugs
(核酸或藥物的定量結合能力分析實驗)

Fluorescence anisotropy

使用螢光光度計(FluoroMax-4/Plus)與偏振片量測樣品螢光各向異性，並算出蛋白與受質解離常數。
We use the fluorometer (FluoroMax-4/Plus) with a polarizer to measure the fluorescence anisotropy to gain the Kd (dissociation constant).

Isothermal Titration Calorimetry (ITC)

以滴定的方式，直接偵測分子相互作用的熱量變化得到熱力學參數：結合親和力（Ka）、焓變（∆H）和結合計量（n）。可用於分析蛋白與蛋白、蛋白與受質、蛋白與藥物間的交互作用。
ITC assay is used to determine the thermodynamic parameters of interactions of the target sample in solution. It can use to analyze the interactions of the protein-protein, protein-substrate, or protein-small molecules.

Intrinsic Tryptophan Fluorescence (ITF)

當蛋白與配體結合時，會改變蛋白內色胺酸的微環境極性，導致色胺酸的螢光訊號發生衰減變化，此現象可用於分析蛋白與配體間的交互作用關係。
The binding between the protein and binding partners affects the local environment polarity around the tryptophan(s) in the protein and results in the intrinsic tryptophan fluorescence quenching. ITF can help us measure the interaction between the protein and binding partners.

Assays for protein-protein complex or multimeric proteins
(蛋白質複合物或蛋白質多聚體測定)

Pull down assay

我們會使用His-tag pull down以及Co-IP來量測複數蛋白質的直接交互作用關係，並會使用Far-western blot分析驗證。
We use His-tag pull-down assay and co-Immunoprecipitation (Co-IP) to detect physical interactions between two or more proteins. Far–western blot analysis is used to confirm.

SEC-MALS

SEC-MALS是一種溶液中絕對分子量測試方法。藉由Size exclusion chromatography將蛋白質樣品分離純化後再利用Multiangle light scattering測定，可以用於分析蛋白質單或多聚體形式，或蛋白質複合體的生成。
Size exclusion chromatography- Multiangle light scattering (SEC-MALS) is used to identify the sample's absolute molecular weight (M.W.) in the solution. After isolation by SEC, we can measure the M.W. of protein in monomer or polymer state and the M.W. of the protein complex.

Crosslink-MASS

Crosslinking assay輔以質譜分析可用於研究測試蛋白質多聚體或異質蛋白質複合體的形成，也可處理蛋白分子樣品來輔助冷凍電子顯微鏡結構測定。
Crosslinking assay combined with Mass spectrometry can measure the formation of protein-polymer or hetero-protein complex. It is also helpful for treating the protein sample for facilitating the structure determination by cryogenic electron microscopy (Cryo-EM).

Assays for protein stability
(蛋白質穩定性測定)

Thermal shift assay (TSA)

記錄當溫度上升時，蛋白質疏水性區域因變性而暴露於外並與螢光染劑產生交互作用時的訊號變化，用於檢測蛋白質在不同實驗條件下的結構穩定程度，如不同成分的緩衝溶液、受質或配體，亦可觀察蛋白質突變對自身結構穩定度的影響。
When the temperature rises, the hydrophobic region of the protein is exposed due to denaturation, and the signal changes when it interacts with the fluorescent dye. TSA is used to detect the structural stability of the protein under different experimental conditions, such as under various buffer solutions, components, substrates, or ligands. The protein mutation on the inherent structural stability of the protein can also be observed.