Nuclear Protein Extraction

Nuclear protein extraction is an important tool in studies on nucleus functions from aspect of proteomics including protein-protein interactions, enzyme activities and post-translational modifications. Determination of the nuclear proteome provides insight into the complex regulatory networks that function in cell nuclei. This procedure usually starts from isolation of pure and enriched nuclei, followed by subsequent extraction of nuclear proteins. Advanced tandem mass spectrometry (MS) is also developed to analyze different types of nuclear proteins, together with their modifications, such as phosphorylation, glycosylation, acetylation and S-nitrosylation.

To isolate the nuclei, a suitable homogenization buffer is first prepared so the integrity of the nuclear membrane and stability of nuclei can be maintained during the isolation from intact cells. Next, isolation of nuclei is processed on ice with sequential steps that include disruption, filtration, centrifugation, solubilization and separation. The separation step by density gradient centrifugation is critical and typically realized through five common methods: (i) differential centrifugation, (ii) discontinuous Percoll gradients, (iii) continuous sucrose gradients, (iv) combined continuous Percoll/sucrose gradients, and (v) continuous Percoll gradients. Different marker proteins can be used to confirm the presence of nuclear proteins or to detect contaminants.

To extract nuclear proteins, a typical protocol is to resuspend the nuclei in Trizol solution first, followed by heating or vortexing to homogenize the sample. Organic solvents such as chloroform are used to separate proteins from DNA and RNA, and gel-based methods or tandem MS technology can be used to evaluate the nuclear protein samples.

Creative BioMart has been the protein expert offering custom services of nuclear protein extraction, powered by years of experience in this field. We prepare the specialized homogenization buffer for various organs and species, select proper detergents to remove membranes from contaminating organelles, and design the most efficient separation methods. With the advanced technology, we generate the most reliable data from nuclear proteomic studies. Creative BioMart offers professional one-stop services for our customer:

• Homogenization buffer preparation.
• Selection of solubilization agents.
• Development of isolation procedure.
• Products evaluation.
• Further mechanisms study.

References:

1. Abdalla, K.O., Thomson, J.A., Rafudeen, M.S. (2009) Protocols for nuclei isolation and nuclear protein extraction from the resurrection plant Xerophyta viscosa for proteomic studies. Analytical Biochemistry. 384: 365-367.
2. Yin, X., Komatsu, S. (2016) Plant nuclear proteomics for unraveling physiological function. New Biotechnology. 33: 644-654.