Simultaneous RNA and protein extraction from the same experimental sample represents a fast-growing demand for researchers who are interested in gene regulation mechanisms. Studies of gene regulation at the mRNA and protein levels are often complicated by small sample sizes and the need to use disparate techniques to obtain nucleic acid and protein. Dividing a sample into several fractions for independent purification procedures is difficult, especially for small samples (e.g., biopsy material). Even more problematic is the difficulty in interpreting experimental results if the samples used for nucleic acid and protein purifications are not identical.
The NucleoSpin RNA/Protein kit allows the concurrent isolation of total RNA and protein from a single sample with no division required, making it especially valuable for unique, small, and/or precious samples. Isolated RNA is suitable for all common downstream applications, with RNA quality and purity comparable to stand-alone RNA purification kits. Isolated protein may be directly used in SDS-PAGE and Western blot analyses.
During the NucleoSpin RNA/Protein procedure, cells are lysed by incubating in lysis buffer containing a large amount of chaotropic ions. The lysis buffer immediately inactivates virtually all enzymes (e.g., RNases, proteases, and phosphatases) that are present in almost all biological materials. The lysis buffer brings even low-solubility proteins into solution and creates appropriate binding conditions to promote the adsorption of RNA to the silica membrane. Under these conditions, protein from all cellular compartments easily passes through the specially treated NucleoSpin RNA/Protein column. The denaturing properties of the lysis buffer eliminate the need for expensive and harmful proteinase inhibitors.
Protein is precipitated from this first flowthrough with the Protein Precipitator (PP) buffer. After a wash step, the protein pellet is dissolved in Protein Solving Buffer (PSB) containing TCEP, an odorless reducing agent. The protein can then be readily used in SDS-PAGE analysis. Contaminating genomic DNA is digested on-column with an RNAse-free recombinant DNase (rDNase, supplied with the kit) which is applied directly to the silica membrane during the workflow. A series of simple washing steps removes salts, metabolites, and macromolecular cellular components. Finally, pure RNA is eluted with RNase-free water.