Introduction The History of Southwestern Blotting Southwestern Blot Mapping Advantages of Southwestern Blotting The Limitations of Southwestern Blotting References
This is because, following the first plotting event, several others have been proposed/discovered to enhance the quality of results. Older protocols were limited by the necessary use of large quantities of proteins and their susceptibility to being degraded while being isolated.
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This technique was first proposed in 1979 and was originally called protein blotting; at the time, existing techniques for purifying proteins associated with DNA had to be combined to produce results. Essentially, Southwestern blotting is modified from a previous procedure called protein blotting, in which proteins were first separated based on size using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the polyacrylate my gel which held the proteins was sandwiched between two filters to cause proteins to diffuse out and bind to produce replicas of the gel.
One of these filters was used to visualize proteins using staining techniques, and the other was used for detecting the protein factors that interacted with the radiolabeled DNA sequence. The older technique typically requires heat inactivation or partial purification of a crude protein extract. Since its inception, several modifications to the initial protocol have been made; namely, electrophoretic transfer of proteins from the gel to the filter is preferred. This enables the renaturation of proteins during the transfer, as the electric field pulls away from the SDS from the proteins.
In addition, an additional modification is an incubation of the filter with milk powder to prevent nonspecific and low-affinity binding sites; this increases the accuracy of the blocked. Finally, salt concentrations are optimized for DNA binding, facilitating high affinity and specific DNA protein interactions.
This form of molecular assay detects specific DNA binding proteins by incubating DNA that has been radiolabeled with a gel blot, washing, and visualizing the products via radiography. Typically, a block is performed using a 1-dimensional SDS page, revealing the binding protein's molecular weight. A two-dimensional gel can be blotted to increase the degree of separation and characterize the isoelectric point. Subsequent dimensions of electrophoresis can be used to further improve separation; one such technique is a gel shift, electrophoretic mobility shift assay (EMSA), which precedes isoelectric focusing and SDS page.
To begin, the proteins under study are prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and consequently loaded onto the gel for separation based on their molecular size. Larger proteins face greater resistance navigating through the mesh-like structure of the gel; in comparison, smaller proteins can fit through the smaller pore sizes. Consequently, large proteins travel less far relative to their smaller counterparts. After time has elapsed, distinct bonds are formed in the gel and located at different positions on the gel compared to the well they were loaded into. These can be visualized using several post-gel electrophoresis staining methods.
Following the protein separation based on molecular weight and size, the gel is electrotransferred (blotted) to a nitrocellulose membrane. A polyvinylidene difluoride membrane may also be used.
To detect the presence of DNA binding protein, the proteins are bound to a nanomolecular concentration of DNA that has been radiolabeled.
Typically, a 32P-labeled double-stranded oligonucleotide probe of a specific DNA sequence is used. Radiolabelled DNA can subsequently bind to the DNA-binding protein to reveal the type of DNA sequence targeted by this specific protein. Any unbound DNA is washed away, and the bands are subsequently screened using autoradiography. This technique is especially useful when identifying the nature of transcription factors because it reveals characteristic information of DNA-binding proteins involved with a particular sequence of DNA.
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The advantage of Southwestern blotting is its ability to provide information regarding the molecular weights of the DNA binding protein. Consequently, this information allows the identity of the protein to be determined using transcription factor databases. Southwestern blotting has been compared to electrophoretic mobility shift assay (EMSA), the most popular method for determining the interaction between transcription factors and DNA.
The principle behind this technique is that the movement of higher molecular weight protein-nucleic acid complexes is more encumbered than the free probe. However, EMSA has several limitations, the most poignant being the binding of a transcription factor to several related DNA sequences.
In addition, the EMSA pattern does not reveal the identity of various proteins present in the complex(es), and subsequent analysis using a super shift assay is necessary. These supershift assays are difficult as selecting the appropriate antibody is challenging and requires prior knowledge to determine the link between the shift pattern and DNA binding protein.
Southwestern blotting requires the separation of proteins under denaturing conditions, which could result in the dissociation of polymeric protein factors, leading to ineffective DNA binding. Consequently, Southwestern blotting is not suited for detecting protein factors that require more than one subunit to bind DNA efficiently.
Moreover, large quantities of DNA are necessary to increase the sensitivity of the SDS-PAGE portion of the technique.
Southwestern blotting aids researchers in understanding the spatial and temporal expression of patterns of the gene, which subsequently enables the elucidation of their function.
Southwestern blotting is particularly useful when investigating DNA protein interactions, particularly those between transcription factors and various regions of DNA periods. The identification of transcription factors that are specific to a gene is significant in understanding both gene regulation and gene function.
Jia Y, Nagore L, Jarrett H. (2015) Southwestern Blotting Assay. Methods Mol Biol. doi:10.1007/978-1-4939-2877-4_5.
Siu FK, Lee LT, Chow BK. (2008) Southwestern blotting in investigating transcriptional regulation. Nat Protoc. doi: 10.1038/nprot.2007.492.