Research And Development Of Spin Column Extraction Technology For Subcellular Structural Proteins

In the post-genomic era,proteomics research is one of the main contents of life science research.With the deepening of research,it has entered the era of subcellular proteomics research.Subcellular structure protein extraction is a necessary step for research,but the traditional subcellular structure protein extraction technology has various defects.Subcellular structure protein extraction is a necessary step for research,but the traditional subcellular structure protein extraction technology has various defects.Such as:the influence of manual homogenization operation on the experiment by human factors,the release of genomic DNA will make the lysate extremely viscous and affect subsequent separation;commonly used surfactants have high cleavage efficiency but may cause protein denaturation and loss;separating subcellular proteins requires high experimental equipment,large sample demand,and so on.This research intends to develop a new column-based protein extraction technology based on a spin column,develop a column-based extraction method and supporting reagents,and develop a corresponding subcellular structure protein separation and extraction kit product.On the basis of the column extraction method,combined with the positively charged SiO₂ nanoparticles on the surface,the cell membrane can be extracted through the charge affinity,and the high-purity extraction of membrane proteins can be achieved.(1)In this study,the microporous structure and surface morphology of the porous material of the centrifugal column filter element formed by pressure sintering were characterized by scanning electron microscope;(2)Carry out total protein extraction and membrane protein extraction,and develop and optimize the basic extraction reagents that match the column extraction method;(3)Based on the basic reagents,target the membrane proteins of cells and tissues,nucleus-cytoplasmic proteins,and mitochondrial proteins,and use commercially available kits as the control group to optimize the basic reagents and extraction schemes,and develop corresponding subcellular structural protein separation and extraction Reagent test kit;(4)Taking the cell membrane as the extraction target,based on the principle of the combination of positive and negative charges,prepared SiO₂ nanoparticles with positive charges on the surface,and characterized them by Zate potentiometer and particle size analysis.After combining the column extraction method,compare the commercially available kits to verify whether it can extract membrane proteins with high purity.The experimental results show that the spin column can effectively lyse cells and remove genomic DNA through centrifugal inertial impact and complex pores.Several subcellular structure protein separation and extraction kits developed have reached the level of excellent commercially available imported kits in terms of extracted protein concentration and protein types,surpassing the currently widely used RIPA extraction method and corresponding kits.The prepared SiO₂ nanoparticles with a positive charge on the surface can be positively charged for a longer time and have a smaller particle size.After being combined with the column method,the membrane protein can be extracted with high purity.Compared with the traditional method,the column extraction method has simple experimental operation,short extraction time,can extract small samples,does not require an ultracentrifuge,and the extraction effect is better than commercially available kits.After the column method is combined with positively charged SiO₂ nanoparticles,membrane proteins can be extracted with high purity.Compared with antibody nanomagnetic beads,it has a lower cost,does not require special magnetic field separation equipment,and is easy to store.Column extraction technology products will effectively reduce equipment requirements,simplify operation steps,and improve extraction results and stability.This technology is expected to be developed into a new scientific research tool and commercialized.Column extraction technology products will effectively reduce equipment requirements,simplify operation steps,and improve extraction results and adaptability.This technology is expected to be developed into a new scientific research tool and commercialized.