A Novel Application for Multiple Form Protein Separation by the Conjugation of Disc Electrophoresis and Isoelectric Focusing: Re-evaluating Cylindrical Disc-PAGE Tube System for Isoelectric Focusing and Potential Outcomes for Future Usage of this Approach

Öz

Aim: Various forms of isoelectric focusing, those involving a single glass column and stabilized by the presence of a sucrose gradient, are preferably used and are the most common pre-application step in 2D electrophoresis. However, the cylindrical disc - PAGE tube system is an exclusive method and has never been evaluated for isoelectric purposes. The goal here was to try a modified application for the separation of some multiple isoform proteins via the multiple tube disc gel system. And to establish isoform separation as a priority and evaluate the implications of this approach for future use. Methods: Essentially, basic features of disc and isoelectric focusing were integrated. Although conventional and therefore the essential component of disc electrophoresis requires three-layers of gel (sample, stacking and separation), only one form gel layer (i.e. separation) was configured, as a first attempt. As a second attempt, the homogenate and ampholite, were deliberately included in the unpolymerised gel medium as if they were gel preparation factors. Then, the separation of the isoforms proteins, which were thought to be homogenously distributed in the presence of ampholite, was achieved. Results: The observation of isoforms of proteins revealed the presence of a distinct pattern of protein bands in their respective isoionic areas. From an analytical perspective, this form of application exhibited very clear and resolved banding patterns. The results obtained regarding the isoforms of hemoglobin proteins are both surprising and informative for us. Conclusions: The banding pattern observed in present approach clearly showed multiple and distinct isoform character profiles. Therefore, this new approach is a clear reflection of the appearance and distinctive power of this novel application. It can be used for in the first dimension of conventional 2D electrophoresis for analytical purposes of multiple proteins separation, when followed vertically.

Anahtar Kelimeler

• IEF
• IPG-Dalt
• Isoform Proteins
• Multiple Hemoglobines
• Dİsk PAGE System

Destekleyen Kurum

No official funding was received for this study.

Etik Beyan

On behalf of all authors, the corresponding author (Naci Değerli) declares that there is no conflict of interest.

Teşekkür

The chemical used in this trial was mostly obtained from supported by the granted MSc Project Thesis Contract No. F-240, and in this way it can be said that this work was partially supported by the Cumhuriyet University Research Fund under MSc Thesis Grant Contract No., F-240.

Disk Elektroforezi ve İzoelektrik Odaklama Konjugasyonu ile Çoklu Protein Formlarını Ayırmak İçin Yeni Bir Uygulama: İzoelektrik Odaklama İçin Silindirik Disk-PAGE Tüp Sisteminin Yeniden Değerlendirilmesi ve Bu Yaklaşımın Gelecekteki Kullanımı İçin Bazı Potansiyel Sonuçları

Öz

Amaç: Bir sukroz gradiyenti varlığında karalı hale getirilmiş tekli cam kolon içeren değişik izoelektrik odaklama yöntemleri, 2D elektroforezin en yaygın biçimi olarak tercihen kullanılır. Ancak, silindirik disk - PAGE tüp sistemi özel bir yöntemdir ve izoelektrik amaçlar için hiç değerlendirilmemiştir. Buradaki amaç, çoklu tüp disk jel sistemi vasıtasıyla bazı çoklu izoform proteinlerinin ayrımı için değiştirilmiş bir uygulamayı denemektir. Ve bir öncelik olarak, izoform ayrımını belirleme yanında, bu yaklaşımın gelecekteki kullanım için olası sonuçlarını değerlendirmektir. Yöntem: Özü itibarıyla, disk ve izoelektrik odaklamanın temel özellikleri entegre edilmiştir. Disk elektroforezinin temel bileşeni üç katmanlı jel (numune ve istifleme ve ayırma) gerektirse de, mevcut girişimde yalnızca ayırma jeli kurgulanmıştır. Basitçe, homojenat ve amfolit, sanki bir jel hazırlama faktörü gibi, polimerize olmamış jel ortamına bilerek dahil edilmiştir. Sonrasında, amfolit varlığında, eşit bir biçimde dağıldığı düşünülen izoform proteinlerin ayrılmasına izin verilmiştir. Genel sonuç: Protein izoformlarının gözlemlenmesi, ilgili izoiyonik alanlarında belirgin bir protein bantları deseninin varlığını ortaya koymuştur. Analitik bir bakış açısından, bu uygulama biçimi çok net, keskin ve ayrışmış bant desenleri sergilemiştir. Dahası, mevcut yaklaşım hemoglobin proteinlerinin izoformları için bazı şaşırtıcı sonuçlara ortaya koymuştur. Sonuç: Bu yaklaşımda gözlemlenen bant deseni, açıkça çoklu ve belirgin izoform karakter profilleri sergilemektedir. Haliyle, bu yeni yaklaşım, bu uygulamanın görünüm ve ayırt edici gücünün açık bir yansımasıdır. Özellikle çoklu protein ayrıştırmalarında, yeri geldiğinde, analitik amaçlar için dikey olanı takiben ilk boyutta kullanılabilir.

Anahtar Kelimeler

• IEF
• IPG-Dalt
• İzoform Proteinler
• Çoklu Hemglobinler
• Disk PAGE Sistemi

Kaynakça

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