2. PRINCIPLE
The principle of northwestern blotting is based on the ability of
RNA-binding proteins to interact specifically with RNA
molecules. These interactions can be detected and
characterized using labeled probes that recognize and bind to
the RNA-binding proteins.
Northwestern blotting is a powerful technique used in the
detection of RNA–protein interaction is used when one wants to
assay the binding of a known RNA to a known or an unknown
protein. In the case of RNA-binding proteins, an RNA probe is
used.
It is worth noting that while Northwestern blotting can detect
RNA-protein interactions, it does not provide information about
the specific RNA sequence(s) to which the protein binds. Other
methods, such as RNA immunoprecipitation (RIP) or RNA
sequencing (RNA-seq), may be used in conjunction with
Northwestern blotting to identify specific RNA targets of RNA-
binding proteins.
3. STEPS IN NORTHWESTERN BLOTTING
1. Sample preparation: The first step is to prepare a sample of RNA-binding proteins and RNA
probe molecules that are believed to interact with each other. The RNA molecules can be either
synthetic or naturally occurring, and may be labeled with radioisotopes, fluorescent dyes, or
biotinylated probes to aid in their detection. The RNA-binding proteins can be obtained from a
variety of sources, including cell lysates, purified proteins, or recombinant proteins.
2. Gel electrophoresis: The RNA-binding proteins separated by gel electrophoresis, usually on a
denaturing polyacrylamide gel, to separate the proteins based on their size. The contaminating
proteins can be separated from the protein of interest on a SDS gel.
3. Protein-RNA transfer: After electrophoresis, the RNA-Binding proteins are transferred to a
nitrocellulose or PVDF membrane using a process called "blotting". During the transfer, the proteins
are immobilized on the membrane in the same relative positions they had on the gel. Staining of the
membrane with Ponceau S stain.
4. Renaturating Buffer: The membrane is submerged in renaturating buffer with gentle shaking.
This is a key step in renaturing or refolding the proteins on the membrane.
5.Blocking: The membrane is then blocked with a non-specific protein, such as bovine serum
albumin (BSA), to prevent non-specific binding of the detection probe.
4. 6.Probe incubation: The membrane is incubated with a labeled probe that recognizes and binds to
the RNA-binding protein. The probe may be a labeled antibody that recognizes the protein directly, or
a labeled RNA or DNA probe that recognizes a specific RNA sequence that is bound by the protein.
The labeled probe is allowed to bind to the RNA-binding protein on the membrane. The amount of
P32 labeled RNA used in the experiment depends on the RNA–protein affinity.
7.Detection: The labeled probe-RNA-protein complexes are then detected using a detection system
appropriate for the type of label used. For example, if the probe is labeled with a radioisotope, the
membrane can be exposed to X-ray film to detect the signal. If the probe is labeled with a fluorescent
dye, the signal can be detected using autoradiography. By estimating the 32P intensity versus the
amount of protein , the Northwestern assay can provide clear evidence that these proteins bind
directly to the RNA activity even when some contaminating RNA-binding protein may exist in the
partially purified protein samples.
