本論文主要是利用微晶片電泳來分析蛋白質間的作用力，實驗的設計主要是讓抗體與抗原能在自由溶液的環境下反應作用，鍵結形成錯合物，再由微晶片電泳的分析，得到兩者形成錯合物的證明。由於人類生理系統中細胞間的訊息傳遞與蛋白質的磷酸化有關，尤其是酪胺酸蛋白激扮演著細胞生長、分化、遷移等重要的角色。因此實驗會先從已純化的磷酸化蛋白質與其抗體的反應作用來分析，進一步分析真實樣品－細胞溶胞產物。整體的實驗架構主要分為二大部份，第一部份先利用毛細管電泳作測試，先分析穩定性高且容易取得的蛋白質，如牛的血清蛋白與其抗體的作用；接下來再分析已純化的磷酸酪胺酸蛋白質。第二部份則是將毛細管電泳的實驗整合於微晶片上，形成利用微晶片電泳分析含磷酸酪胺酸的胜或蛋白質與其抗體的作用。實驗中所採用的是十字形的玻璃晶片，利用電壓的切換與時間的調控，達到樣品的注射與分離。抗體與抗原的反應，通常配合抗體不同濃度的滴定，造成圖譜中波峰的改變，並嘗試從圖譜中未鍵結型態的抗原與錯合物兩者波峰面積的改變，推算出鍵結常數。實驗結果顯示，含磷酸酪胺酸胜（FITC-peptide(pTyr)）與含磷酸酪胺酸蛋白質（pTyr-BSA）分別在不同濃度的磷酸酪胺酸抗體（anti-P）反應作用下，與不含磷酸酪胺酸胜之對照組比對，可以明顯判斷出未鍵結型態與錯合物的波峰差異，並由波峰面積的改變，我們分別推算出鍵結常數K=5.0×106 M-1與K=1.01×106 M-1。
在真實樣品的分析中，由於細胞溶胞產物所含的蛋白質種類不只一種，對於利用磷酸酪胺酸的抗體來辨識其抗原，容易產生基質干擾的問題，所以我們先對基質影響進行相關實驗，利用染料標示含磷酸酪胺酸的蛋白質（Cy5-pTyr-BSA）與不同比率的細胞溶胞產物（由phoenix細胞轉染二個質體，即pcDNA3.0-T7-Etk和pRL-c-SrcY527F）混合。由實驗結果發現真實樣品中含有強的BSA鍵結蛋白質。我們進一步利用FITC-anti-P對不同濃度的細胞溶胞產物反應作用，以偵測其含之磷酸化蛋白質，所得到的結果顯示有明顯的波峰形狀改變，但卻難以分辨抗體與抗體錯合物之訊號，這一部份的實驗仍待進一步的驗證。
微晶片電泳不論在實驗時間與試劑用量都較傳統免疫分析法及毛細管電泳少，而且因為微管道的長度較短，單位電場強度可以較低，分析時間可以大為縮短，因此不容易破壞抗體與抗原的鍵結，有利於快速篩選及分析蛋白質間的作用力。

In this thesis, we investigated protein-protein interaction using microchip electrophoresis. In this design, the Cy5-labelled antigen was first incubated with the antibody to form the interaction complex in the free solution and then separated from the complex species by microchip electrophoresis. The evidence of forming the interaction complex was based on the detection of both the free and the complex species and the variation of the related the peak intensity by immuno-titration. The protein phosphorylation plays an important role in regulating cellular growth, differentiation, and migration, the experiment was designed to detect the protein phosphorylation using anti phosphorylation antibody as the affinity bite.
There are two major sections in the experiment. In the first section, we used the capillary electrophoresis to analyze a model system—anti-BSA and BSA and then analyze the target goal—phospho peptides and anti-phosphotyrosine. In the second section, we transferred the experiment from capillary electrophoresis to the microchip. In microchip electrophoresis, the sample injection and separation was integrated in a cross-microchannel which was etched on the glass substrate. The binding constant of the phospho peptide/phospho protein and the antibody was estimated by immuno-titration in which the concentration ratio between the antigen and the antibody was systematically varied. The result indicated that both the free form and complex form of comparing the FITC-peptide(pTyr) or Cy5-pTyr-BSA can be clearly detected and confirmed by the comparison with results obtained using a non-phosphorylated peptide or protein counterpart as the negative control. The binding constants for FITC-peptide(pTyr) or Cy5-pTyr-BSA with anti-phosphotyrosine, respectively was estimated to be 5.0×106 M-1 and 1.01×106 M-1.
For real sample analysis, we first investigated the matrix effect by spiking Cy5-pTyr-BSA into the cell lysate (transfection of the phoenix cell using two plasmid, pcDNA3.0-T7-Etk and pRL-c-SrcY527F) at various ratios. From the experiment, we found that the real sample may contain the strong BSA binding proteins. We further used FITC-anti-phosphotyrosine to react with cell lysate at different concentrations to detect the phospho-protein which was supposed to be present in the cell lysate. An obvious change of the peak shape for both the free and complex species was noticed. It was however difficult to differentiate the signal of the antibody and the complex, which requires further investigations.
Compared to the traditional immunoassay and capillary electrophoresis, the experiment time and the amount of the reagent can be greatly reduced using microchip electrophoresis w. Moreover, because of the shorter microchannel than capillary electrophoresis such that the interaction complex can be conserved and the analysis time was much shorter. Therefore, microchip electrophoresis is potentially capable of high-throughput screening of protein-protein interaction before the detection.

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