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[[File:Dot blot de ADN.jpg|thumb|Typical dot blot membrane. Darker dots indicate more protein.]] |
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{{No footnotes|date=October 2014}} |
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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. |
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[[image:DotBlotDemo.jpg|right|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate dot blot filters.]] |
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A '''dot blot''' (or '''slot blot''') is a technique in [[molecular biology]] used to detect [[biomolecule]]s, and for detecting, analyzing, and identifying proteins. It represents a simplification of the [[northern blot]], [[Southern blot]], or [[western blot]] methods. In a dot blot the biomolecules to be detected are not first separated by [[electrophoresis]]. Instead, a mixture containing the [[molecule]] to be detected is applied directly on a membrane as a dot, and then is spotted through circular templates directly onto the membrane or paper substrate. This differs from the [[western blot]] because protein samples are not separated electrophoretically. This is then followed by detection by either [[nucleotide]] [[hybridization probe|probes]] (for a [[northern blot]] and [[southern blot]]) or [[antibody|antibodies]] (for a [[western blot]]). |
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The technique offers significant savings in time, as [[chromatography]] or [[gel electrophoresis]], and the complex blotting procedures for the gel are not required. |
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.<ref name="biorad">{{cite web|title=Test Blots, Slot Blots & Dot Blots - Immunodetection {{!}} Bio-Rad|url=https://www.bio-rad-antibodies.com/immunodetection-test-blots-slot-blots-dot-blots-western-blotting.html|website=Bio-Rad|language=en}}</ref> |
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== Uses == |
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Because a dot blot does not require any specific instruments, lots of dot blot assays are developed using antibodies with high specificity to detect different protein targets. Dot blot is also used to evaluate or screen the effectiveness of the antibodies. |
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Performing a dot blot is similar in idea to performing a western blot, with the advantage of faster speed and lower cost. |
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== Protocol== |
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The following is a general outline of an antigen-antibody-antibody dot blot assay protocol. Specific concentrations need to be determined for each assay. Other types of dot blot assays, such as antibody-antigen-antibody can be performed in a similar fashion. |
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Dot blots are also performed to screen the binding capabilities of an antibody.<ref>{{cite journal|last1=Rupprecht|first1=Kevin R.|last2=Nair|first2=Rad K.|last3=Harwick|first3=Larissa C.|last4=Grote|first4=Jonathan|last5=Beligere|first5=Gangamani S.|last6=Rege|first6=Sushil D.|last7=Chen|first7=Yon-Yih|last8=Lin|first8=Zhen|last9=Fishpaugh|first9=Jeffrey R.|title=Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs|pmid=20696124|journal=Analytical Biochemistry|pages=160–164|doi=10.1016/j.ab.2010.08.003|date=15 December 2010|volume=407 |issue=2 }}</ref> |
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1. spot 1-2 microliters of antigen on to a piece of membrane, let air dry for 30 min or longer; |
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2. incubate with blocking buffer for 30 min -2 hr; |
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3. rinse with rinsing buffer, 3x5 min; |
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4. incubate with primary antibody, 30 min - 2 hr; |
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5. rinse with rinsing buffer, 3x5 min; |
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6. incubate with enzyme-labeled secondary antibody, 30 min - 2 hr; |
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7. rinse with rinsing buffer, 3x5 min; |
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8. add enzyme substrate, wait 5-10 min; |
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9. detect by eye or with colorimetric or chemiluminescent imaging system. |
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== Methods == |
== Methods == |
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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.<ref name="abcam">{{cite web|url=http://www.abcam.com/ps/pdf/protocols/dot%20blot%20protocol.pdf|title=Dot Blot Protocol}}</ref> |
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| ⚫ | |||
The membrane is incubated in blocking buffer to prevent non-specific binding of antibodies. It is then incubated with a primary antibody followed by a detection antibody or a primary antibody conjugated to a detection molecule (commonly [[horseradish peroxidase|HRP]] or [[alkaline phosphatase]]). After antibody binding, the membrane is incubated with a [[chemiluminescent]] substrate and imaged. |
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| ⚫ | |||
== Apparatus == |
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Vitrozm's 96 well Zoom Blot method uses an absorption plug to wick away the solution from the membranes, which are individually assembled in each well. The Zoom Plate is a disposable self-standing device, without any external vacuum or set up apparatus, which allows dot blots to be performed much easier and faster. The user never needs to handle a piece of membrane as in older methods. Zoom Blot's 96 well plate format allows convenient signal detection and quantification using 96 well plate reader or imaging system. |
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| ⚫ | 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. |
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| ⚫ | 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 disassembled and the membrane need to be taken out and wrapped in a transparent plastic film. |
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== Dot blot Detection Capabilities == |
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The sensitive dot blot test can be used to detect the ''[[Chlamydia trachomatis]]'' infection and |
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other [[sexually transmitted diseases]]. The dot blot is used to detect Antidiacyltrehalose [[Antibodies]] in [[Tuberculous]] patients and [[Typhoid Fever]]. |
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== See also == |
== See also == |
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* [[ |
* [[ELISpot]] |
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* [[Southern blot]] |
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* [[Northern blot]] |
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* [[Western blot]] |
* [[Western blot]] |
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* [[Southwestern blot]] |
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== External links == |
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*[http://www.bio-rad.com/cmc_upload/Literature/12484/M1706542C.pdf A protocol for the BioRad Dot-Blot Apparatus]{{dead link|date=December 2016 |bot=InternetArchiveBot |fix-attempted=yes }} |
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*[http://www.vitrozm.com/pages/zoom-blot-fast-and-easy-96-well-dot-blot Dot Blot using a Zoom Plate] |
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== References == |
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== Citations and More Information == |
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{{reflist}} |
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*[http://cvi.asm.org/content/7/2/312.full] |
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*[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95755/] |
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*[http://www.abcam.com/ps/pdf/protocols/Dot%20blot%20protocol.pdf] |
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*[http://www.motherlandinc.org/clinic/index.php?option=com_content&task=view&id=16&Itemid=9]{{dead link|date=December 2016 |bot=InternetArchiveBot |fix-attempted=yes }} |
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*[http://www.biosynth.com/index.asp?topic_id=144]{{dead link|date=December 2016 |bot=InternetArchiveBot |fix-attempted=yes }} |
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[[Category: |
[[Category:Genetics techniques]] |
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[[Category:Molecular biology techniques]] |
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Revision as of 03:43, 23 March 2024
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]
Uses
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
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 of antibodies. 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
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 disassembled and the membrane need to be taken out and wrapped in a transparent plastic film.
See also
References
- ^ "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. 407 (2): 160–164. doi:10.1016/j.ab.2010.08.003. PMID 20696124.
- ^ "Dot Blot Protocol" (PDF).