Northwestern blot

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The northwestern blot, also known as the northwestern assay, is a hybrid analytical technique of the western blot and the northern blot, and is used in molecular biology to detect interactions between RNA and proteins. A related technique, the western blot, is used to detect a protein of interest that involves transferring proteins that are separated by gel electrophoresis onto a nitrocellulose membrane. A colored precipitate clusters along the band on the membrane containing a particular target protein. A northern blot is a similar analytical technique that, instead of detecting a protein of interest, is used to study gene expression by detection of RNA (or isolated mRNA) on a similar membrane. The northwestern blot combines the two techniques, and specifically involves the identification of labeled RNA that interact with proteins that are immobilized on a similar nitrocellulose membrane.

Contents

History

Edwin Southern first created the Southern blot, [1] an analytical technique used to detect DNA. The technique involves using gel electrophoresis, an important analytical method that involves the use of an electric field and the subsequent migration of charged DNA, RNA or proteins through that electric field based on size and charge. [2] With a Southern Blot, the separated DNA fragments are then transferred to a filter membrane for detection. [1] Detection occurs as bands become visible on the membrane and correlate with a particular molecule of interest. [2] Subsequently, other similar blotting techniques were created with similar nomenclature to detect different molecules or interactions between molecules. These techniques include the western blot (protein detection), the northern blot (RNA detection), the southwestern blot (DNA-protein interaction detection), the eastern blot (post translational modification detection) and the northwestern blot (RNA-protein interaction detection). [3] [4] [5] [6]

Technique specifics

Running a northwestern blot involves separating the RNA binding proteins by gel electrophoresis, which will separate the RNA binding proteins based upon their size and charge. Individual samples can be loaded in to the agarose or polyacrylamide gel (usually an SDS-PAGE) in order to analyze multiple samples at the same time. [5] Once the gel electrophoresis is complete, the gel and associated RNA binding proteins are transferred to a nitrocellulose transfer paper. [7]

The newly transferred blots are then soaked in a blocking solution; non-fat milk and bovine serum albumin are common blocking buffers. [8] This blocking solution assists with preventing non-specific binding of the primary and/or secondary antibodies to the nitrocellulose membrane. Once the blocking solution has adequate contact time with the blot, a specific competitor RNA is applied and given time to incubate at room temperature. During this time, the competitor RNA binds to the RNA binding proteins in the samples that are on the blot. The incubation time during this process can vary depending on the concentration of the competitor RNA applied; though incubation time is typically one hour. [9] After the incubation is complete, the blot is usually washed at least 3 times for 5 minutes each wash, in order to dilute out the RNA in the solution. Common wash buffers include Phosphate buffered saline (PBS) or a 10% Tween 20 solution. [10] Improper or inadequate washing will affect the clarity of the development of the blot. Once washing is complete the blot is then typically developed by x-ray or similar autoradiography methods. [11]

Applications

After developing the blot using xray or autoradiography, the results can be analyzed and interpreted to determine the approximate size and concentration of the RNA binding protein(s) of interest to further study the protein(s). The location and concentration of the RNA binding protein on the blot can affect the results, and bands can sometimes appear after development. These bands can help researchers determine the size and concentration of the RNA binding protein of interest. [12] When the approximate size of the protein is known, the original sample can be run on a chromatography machine to separate it by size. [13] In addition, once the protein is isolated, it can be digested with trypsin, and Mass Spectrometry can be utilized to sequence the peptides in order to determine the identity of the specific protein. [14]

Advantages and disadvantages

Advantages of northwestern blotting include the expedited detection of specific proteins that bind RNA, as well as the assessment of the approximate molecular weights of those proteins. [15] The northwestern blot allows for detection of identified proteins in a way that is inexpensive. The blot is typically a first step in research, as it allows for the identification of the approximate molecular weights, once the molecular weight is known it allows for further research or purification through other methods like chromatography. Another advantage of the northwestern blot is that it aides in the building of expression libraries of cognate ligands. [16]

A noted disadvantage is that some RNA-Protein interactions with poor RNA binding properties may not be as detectable with this technique. [15] Also the procedure for blotting can take from 3 to 5 hours. If the procedure is not done correctly it can result in significant background which can result in an unclear blot of the proteins identified. In addition, proteins need to renature after being separated and transferred to the nitrocellulose membrane. One last disadvantage is that proteins must consist of a single polypeptide or two subunits that comigrate in the gel matrix. [17]

See also

Protocols

Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles

RNA Isolation and Northern Blot Analysis

Protein Blotting

Related Research Articles

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Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physical structure of biological macromolecules is known as molecular biology.

<span class="mw-page-title-main">Northern blot</span> Molecular biology technique

The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA in a sample.

<span class="mw-page-title-main">Southern blot</span> DNA analysis technique

Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample is digested with restriction enzymes, and the resulting DNA fragments are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.

<span class="mw-page-title-main">Polyacrylamide gel electrophoresis</span>

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.

<span class="mw-page-title-main">Western blot</span> Analytical technique used in molecular biology

The western blot, or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.

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<span class="mw-page-title-main">Blot (biology)</span>

A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA onto a carrier. In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining, autoradiographic visualization of radiolabelled molecules, or specific labelling of some proteins or nucleic acids. The latter is done with antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity is visualized by incubation with proper reactive, rendering either a colored deposit on the blot or a chemiluminescent reaction which is registered by photographic film.

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<span class="mw-page-title-main">Southwestern blot</span> Molecular biology technique

The southwestern blot, is a lab technique that involves identifying as well as characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes. Determination of molecular weight of proteins binding to DNA is also made possible by the technique. The name originates from a combination of ideas underlying Southern blotting and Western blotting techniques of which they detect DNA and protein respectively. Similar to other types of blotting, proteins are separated by SDS-PAGE and are subsequently transferred to nitrocellulose membranes. Thereafter southwestern blotting begins to vary with regards to procedure as since the first blotting’s, many more have been proposed and discovered with goals of enhancing results. Former protocols were hampered by the need for large amounts of proteins and their susceptibility to degradation while being isolated.

<span class="mw-page-title-main">Electrophoretic mobility shift assay</span>

An electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common affinity electrophoresis technique used to study protein–DNA or protein–RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation, DNA gang replication, DNA repair or RNA processing and maturation, as well as pre-mRNA splicing. Although precursors can be found in earlier literature, most current assays are based on methods described by Garner and Revzin and Fried and Crothers.

<span class="mw-page-title-main">Dot blot</span>

A dot blot is a technique in molecular biology used to detect proteins. It represents a simplification of the western blot method, with the exception that the proteins to be detected are not first separated by electrophoresis. Instead, the sample is applied directly on a membrane in a single spot, and the blotting procedure is performed.

The far-western blot, or far-western blotting, is a molecular biological method based on the technique of western blot to detect protein-protein interaction in vitro. Whereas western blot uses an antibody probe to detect a protein of interest, far-western blot uses a non-antibody probe which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is employed to detect protein/protein interactions.

<span class="mw-page-title-main">Molecular-weight size marker</span> Set of standards

A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.

<span class="mw-page-title-main">Affinity electrophoresis</span>

Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. Cross electrophoresis, the first affinity electrophoresis method, was created by Nakamura et al. Enzyme-substrate complexes have been detected using cross electrophoresis. The methods include the so-called electrophoretic mobility shift assay, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one-dimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to affinity chromatography by use of immobilized ligands.

The eastern blot, or eastern blotting, is a biochemical technique used to analyze protein post-translational modifications including the addition of lipids, phosphates, and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thus, eastern blot can be considered an extension of the biochemical technique of western blot. Multiple techniques have been described by the term "eastern blot(ting)", most use phosphoprotein blotted from sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel on to a polyvinylidene fluoride or nitrocellulose membrane. Transferred proteins are analyzed for post-translational modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification. Eastern blotting should be used to refer to methods that detect their targets through specific interaction of the post-translational modifications and the probe, distinguishing them from a standard far-western blot. In principle, eastern blotting is similar to lectin blotting.

A blotting matrix, in molecular biology and genetics, is the substrate onto which macromolecules, such as proteins, are transferred in a blot method. The matrices are generally chemically modified paper filters or microporous membrane filters. In a dot blot, macromolecules are applied directly to the matrix. Macromolecules can also be separated and transferred via gel electrophoresis.

<span class="mw-page-title-main">SDS-PAGE</span> Biochemical technique

SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate and polyacrylamide gel allows to eliminate the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight. At least up to 2012, the publication describing it was the most frequently cited paper by a single author, and the second most cited overall.

References

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  2. 1 2 Nelson, Cox (2013). Lehninger Principles of Biochemistry. New York, NY: W.H. Freeman and Company. p. 179. ISBN   978-1-4641-0962-1.
  3. ↑ Alberts, B., Johnson, A., Lewis, J. Raff, M., Roberts, K., Walter, P. 2008. Molecular Biology of the Cell, 5th ed. Garland Science, Taylor & Francis Group, NY, pp 538-539.
  4. ↑ Kevil, C. G., Walsh, L., Laroux, F. S., Kalogeris, T., Grisham, M. B., Alexander, J. S. (1997) An Improved, Rapid Northern Protocol. Biochem. and Biophys. Research Comm. 238:277-279.
  5. 1 2 Rapley, R (2000). The Nucleic Acid Protocols Handbook. Totowa, NJ: Humana Press Inc. p. 783. ISBN   978-0-89603-459-4.
  6. ↑ Nicholas; Nelson (2013). "North, South, or East? Blotting Techniques". Journal of Investigative Dermatology. 133 (e10): e10. doi: 10.1038/jid.2013.216 . PMID   23760052.
  7. ↑ Verena, Bichsel; Alfred Walz; Matthias Bickel (1997). "Identification of proteins binding specifically to the 3'-untranslated region of granulocyte/macrophage-colony stimulating factro mRNA". Nucleic Acids Research. 25 (12): 2417–2423. doi:10.1093/nar/25.12.2417. PMC   146745 . PMID   9171094.
  8. ↑ Liao, Huey-Jane; Ryuji Kobyashi and Michael B. Matthews; Mathews, M. B. (July 21, 1998). "Activities of adenovirus virus-associated RNAs: Purification and characterization of RNA binding proteins". Proceedings of the National Academy of Sciences of the United States of America. 95 (15): 8514–9. Bibcode:1998PNAS...95.8514L. doi: 10.1073/pnas.95.15.8514 . PMC   21107 . PMID   9671709.
  9. ↑ C, Franke; Grafe D; Bartsch H; Bachmann M (2009). Use of non-radioactive detection method for north- and southwestern blot. Methods in Molecular Biology. Vol. 536. pp. 441–9. doi:10.1007/978-1-59745-542-8_44. ISBN   978-1-934115-73-2. PMID   19378081.
  10. ↑ Schumacher, Jill; Keesook Lee; Susanne Edelhoff; Robert Braun (May 1995). "Spnr, a Murine RNA-binding Protein That Is Localized to Cytoplasmic Microtubules". The Journal of Cell Biology. 129 (4): 1023–1032. doi:10.1083/jcb.129.4.1023. PMC   2120489 . PMID   7744952.
  11. ↑ Stohlman, S A; R S Baric; G N Nelson; L H Soe; L M Welter; R J Deans (1988). "Specific interaction between coronavirus leader RNA and nucleocapsid protein". Journal of Virology. 11. 62 (11): 4288–95. doi:10.1128/JVI.62.11.4288-4295.1988. PMC   253863 . PMID   2845141.
  12. ↑ Thangasamy, Saminathan (5 April 2013). "Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles". Bio-Protocol. 3 (7): e625. doi:10.21769/BioProtoc.625 . Retrieved 26 March 2014.
  13. ↑ Perdew, Gary (Aug 17, 2008). Regulation of Gene Expression: Molecular Mechanisms. Springer. p. 129. ISBN   9781597452281.
  14. ↑ Gary H. Perdew; Jack P. Vanden Heuvel; Jeffrey M. Peters (2008). Regulation of Gene Expression: Molecular Mechanisms. Springer. p. 129. ISBN   9781597452281.
  15. 1 2 Smith, Christopher W.J. (1998). RNA-Protein Interactions : A Practical Approach: A Practical Approach. Oxford University Press. p. 187. ISBN   9780191591624.
  16. ↑ Waldo, Cohen (Aug 16, 1991). Progress in Nucleic Acid Research and Molecular Biology. Academic Press. p. 186. ISBN   9780080863290.
  17. ↑ Nicholson, Allen W. (2001). Ribonucleases, Part A: Functional Roles and Mechanisms of Action: Functional Roles and Mechanisms of Action. Academic Press. p. 409. ISBN   9780080496917.
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