A comparison of protein extraction methods suitable for gel-based proteomic studies of spider silk proteins

In proteomic studies of silk biomaterial, a reliable protein extraction method is critical. Various methods have been published, yet very few works on comparing them. Here, we evaluate the effect of three different methods on Nephila pilipes‘s spider silk fibre structure, its protein yield and separ...

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Bibliographic Details
Main Authors: Abdullah, N. A. H., Mohtar, J. A., Ismail, K. S. Ku, Khoo, B. Y.
Format: Conference Proceeding
Language:English
Subjects:
Biomedical materials
Electrophoresis
Fibers
Lithium
Physical properties
Polyacrylamide
Proteins
Silk
Sodium dodecyl sulfate
Spiders
Thiocyanates
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Summary:In proteomic studies of silk biomaterial, a reliable protein extraction method is critical. Various methods have been published, yet very few works on comparing them. Here, we evaluate the effect of three different methods on Nephila pilipes‘s spider silk fibre structure, its protein yield and separation on polyacrylamide gel. The major ampullate spider silk fibres were solubilized using urea, lithium thiocyanate, or lithium bromide solution in order to extract the major ampullate spidroin proteins. Our results revealed that the 9.3M lithium bromide (LiBr) solution was the most effective dissolving agent for Nephila pilipes‘s spider silk fibres. The two most prominent major spidroin proteins were successfully separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) while microscopic visualization on Scanning Electron Microscope (SEM) reveals the immediate deformation of silk fibres. Throughout the analysis, it was due to the suitable concentration (ionic strength of solution) and acidic content of the denaturant that gave the greatest and immediate impact on unfolding the highly stable, hydrophobic, insoluble, and aggregation-prone spidroins. While other physical parameters such as temperature as well as agitation, they enhance the spidroin protein extraction time.
ISSN:0094-243X
1551-7616
DOI:10.1063/5.0046063