Principle

Parallel isolation of RNA and protein from undivided samples

Simultaneous RNA and protein extraction from the same experimental sample is a great and fast growing demand for researchers interested in gene regulation mechanisms. Studies of gene regulation at the level of mRNA as well as cognate protein are often complicated by small sample sizes and the need to use differing and often incompatible techniques to obtain DNA, RNA, and protein. Dividing a sample into several parts for independent purification procedures is difficult especially for small samples (e.g., biopsy material). Even more problematic is the difficulty in interpretation of experimental results as the analysis might be mistrusted if the experimental sample for nucleic acid and protein purification is not absolutely identical. The NucleoSpin® RNA/Protein kit allows for the concurrent isolation of RNA and total cellular protein from undivided samples in one working procedure. Protein and RNA are obtained in separate fractions. Furthermore, in combination with a separately available buffer set (REF 740944) the simultaneous purification of DNA is possible. Protein and RNA are isolated without splitting the sample prior to extraction. Thus, protein and RNA are obtained from one and the same entire sample. This is especially valuable for unique, small, and precious samples. Isolated RNA is suitable for all common downstream applications and is of identical high quality as RNA isolated with the well proven NucleoSpin® RNA II kit. Isolated protein is immediately suitable for SDS-PAGE and Western blot analysis.

During the NucleoSpin® RNA/Protein procedure, cells are lysed by incubation in a solution containing large amounts of chaotropic ions. This lysis buffer immediately inactivates virtually all enzymes (e.g., RNases, proteases, and phospatases) which are present in almost all biological materials. The buffer dissolves even hardly soluble proteins and creates appropriate binding conditions which favor adsorption of RNA to the silica membrane upon addition of the lysate to the column. Under these conditions protein from all cellular compartments is enabled to pass the specially treated NucleoSpin® RNA/Protein column virtually quantitatively. The denaturing properties of the lysis buffer make expensive and harmful proteinase inhibitors or inhibitor cocktails unnecessary. Protein is precipitated from the first column flow-through with a special buffer (Protein Precipitator, PP). After a washing step the protein pellet is dissolved in Protein Solving Buffer (PSB) containing the odourless reducing agent TCEP. The protein can thus readily be used in SDS-PAGE analysis. Contaminating genomic DNA is digested on-column with a recombinant DNase (rDNase) which is directly applied to the silica membrane during the preparation (RNase-free rDNase is supplied with the kit). Simple washing steps with two different buffers remove salts, metabolites, and macromolecular cellular components. Pure RNA is finally eluted with RNase-free water under low ionic strength conditions.

The RNA and protein preparation using NucleoSpin® RNA/Protein can be performed at room temperature. The RNA eluate should be treated with care as it is common practice for handling of RNA. The NucleoSpin® RNA/DNA Buffer Set is a support set for additional DNA isolation. The combination with NucleoSpin® RNA/Protein allows parallel isolation of RNA, DNA, and protein from one undivided sample. This patented technology enables successive elution of DNA and RNA from one NucleoSpin® column with low salt buffer and water, respectively. DNA and RNA are immediately ready for downstream applications.

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