Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method | Tian

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Typical dot blot membrane. Darker dots indicate more protein.

A dot blot (or slot 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 technique offers significant savings in time, as chromatography or gel electrophoresis, and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target protein.^[1]

Dot and slot blots differ only in the geometry of the blot, a series of spots giving a hybridization pattern that is amenable to analysis by densitometric scanning. Samples are usually applied to.

As an alternative, Dot blot analysis was developed to simplify the process of Western blot analysis. In fact, both enzyme-linked immunosorbent assay (ELISA) 6-8 and reverse phase protein microarray (RPPM) 9, 10 can be considered as Dot Blot analysis in a high throughput format. Nonetheless, the applications of these techniques are still.
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Uses[edit]

Performing a dot blot is similar in idea to performing a western blot, with the advantage of faster speed and lower cost.

Dot blots are also performed to screen the binding capabilities of an antibody.^[2]

Methods[edit]

A general dot blot protocol involves spotting 1-2 microliters of a samples onto a nitrocellulose or PVDF membrane and letting it air dry. Samples can be in the form of tissue culture supernatants, blood serum, cell extracts, or other preparations.^[3]

The membrane is incubated in blocking buffer to prevent non-specific binding. It is then incubated with a primary antibody followed by a detection antibody or a primary antibody conjugated to a detection molecule (commonly HRP or alkaline phosphatase). After antibody binding, the membrane is incubated with a chemiluminescent substrate and imaged.

Apparatus[edit]

Dot blot is conventionally performed on a piece of nitrocellulose membrane or PVDF membrane. After the protein samples are spotted onto the membrane, the membrane is placed in a plastic container and sequentially incubated in blocking buffer, antibody solutions, or rinsing buffer on shaker. Finally, for chemiluminescence imaging, the piece of membrane need to be wrapped in a transparent plastic film filled with enzyme substrate.

Vacuum-assisted dot blot apparatus has been used to facilitate the rinsing and incubating process by using vacuum to extract the solution from underneath the membrane, which is assembled in between several layers of plates to ensure good seal between sample wells, hold waste solution, and deliver suction force. For chemiluminescence signal detection, apparatus need to be dissembled and the membrane need to be taken out and wrapped in a transparent plastic film.

References[edit]

^'Test Blots, Slot Blots & Dot Blots - Immunodetection | Bio-Rad'. Bio-Rad.
^Rupprecht, Kevin R.; Nair, Rad K.; Harwick, Larissa C.; Grote, Jonathan; Beligere, Gangamani S.; Rege, Sushil D.; Chen, Yon-Yih; Lin, Zhen; Fishpaugh, Jeffrey R. (15 December 2010). 'Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs'. Analytical Biochemistry: 160–164. doi:10.1016/j.ab.2010.08.003. PMID20696124.
^'Dot Blot Protocol'(PDF).

Retrieved from 'https://en.wikipedia.org/w/index.php?title=Dot_blot&oldid=928136135'

Research Papers:

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Oncotarget. 2017; 8:58553-58562. https://doi.org/10.18632/oncotarget.17236

Abstract

Geng Tian1,*, Fangrong Tang2,*, Chunhua Yang1, Wenfeng Zhang2, Jonas Bergquist3, Bin Wang1, Jia Mi1,3 and Jiandi Zhang1,2

1Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai, P. R. China

2Yantai Zestern Biotechnique Co. LTD, Yantai, P. R. China

3Department of Chemistry, BMC, Uppsala University, Uppsala, Sweden

*These authors have contributed equally to this work

Correspondence to:

Jiandi Zhang, email: Jiandi.zhang@zestern.net

Jia Mi, email: jiajiami@gmail.com

Keywords: quantitative dot blot, high throughput, proteomics, immunoblot analysis, western blot

Received: February 17, 2017 Accepted: March 26, 2017 Published: April 19, 2017

ABSTRACT

Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this “Big Data” era.

Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method | Tian | Oncotarget

INTRODUCTION

Lacking of an accessible high throughput immun-oblot method significantly hinders any attempts to investigate the molecular basis of biological and pathological processes systematically in an average research lab. Until now, Western blot analysis remains the most commonly used immunoblot tool in the basic research lab almost 40 years since its invention [1-4]. However, its limitations, including complicated processing steps, limited ability to process many samples, ambiguity in result analysis and requirement of large amount of total protein lysate for analysis, determines this technique an unlike choice for high throughput analysis. As an alternative, Dot blot analysis was developed to simplify the process of Western blot analysis [5]. In fact, both enzyme-linked immunosorbent assay (ELISA) [6-8] and reverse phase protein microarray (RPPM) [9, 10] can be considered as Dot Blot analysis in a high throughput format. Nonetheless, the applications of these techniques are still limited in basic research lab for various reasons, including limited availability for prefabricated ELISA kits and lack of easy access to RPPMs.

Meanwhile, with the rapid development of effective high-throughput tools in genetic research, there are strong demands for complementary high throughput immunoblot methods on a daily basis for biomarker identification and other association studies at protein level [11, 12]. We have now developed a novel immunoblot method as convenient and robust as the traditional dot blot

Slot And Dot Blot Methodologies