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{{Short description|Technique for protein detection}}
[[image:DotBlotDemo.jpg|right|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate dot blot filters.]]
[[File:Dot blot de ADN.jpg|thumb|Typical dot blot membrane. Darker dots indicate more protein.]]
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 [[biomolecule]]s to be detected are not first separated by [[chromatography]]. 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]]).
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 [[biomolecule]]protein.<ref name="biorad">{{cite Furthermore,web if|title=Western twoBlot: [[molecule]]sTest of different sizesBlots, areSlot detectedBlots, theyand willDot still appear as a singleBlots |url=https://www.bio-rad-antibodies.com/immunodetection-test-blots-slot-blots-dot-blots-western-blotting.html |website=Bio-Rad Dot|access-date=8 blotsAugust therefore2024 can only confirm the presence or absence of a [[biomolecule]] or [[biomolecule]]s which can be detected by the DNA [[hybridization probe|probes]] or the [[antibody]].language=en}}</ref>
 
== Uses ==
A radioactive sample can be [[nucleic acid hybridisation|hybridized]] to allow for the detection of variation between samples. The DNA is quantified and equal amounts are aliquoted into tubes in excess of the number of its targets in the samples, such as 10&nbsp;µg for a plasmid and 1&nbsp;µg for a PCR amplicon. These are denatured (NaOH and 95°C) and added to the wells where a vacuum sucks the water (with NaOH and NH<sub>4</sub>OAc) from underneath the membrane (nylon or nitrocellulose).
Performing a dot blot is similar in idea to performing a western blot, with the advantage of faster speed and lower cost.<ref>{{cite journal |last1=Cheng |first1=Li-Ting |last2=Zeng |first2=Yu-Jing |last3=Chu |first3=Chun-Yen |last4=Wang |first4=Hsian-Yu |title=Development of a quick dot blot assay for the titering of bovine ephemeral fever virus |journal=BMC Veterinary Research |date=December 2019 |volume=15 |issue=1 |doi=10.1186/s12917-019-2059-6|doi-access=free |pmid=31477093 |pmc=6720828 }}</ref> 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>
 
== Methodology ==
== What can this test detect? ==
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 |title=Dot blot protocol |url=https://www.abcam.com/en-us/technical-resources/protocols/dot-blot |website=abcam |access-date=8 August 2024 |date=21 May 2023}}</ref> 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. After antibody binding, the membrane is incubated with a [[chemiluminescent]] substrate and imaged.
 
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.
The sensitive dot blot test can be used to detect the [[Chlamydia trachomatis infection]] and
other [[sexually transmitted diseases]]. Dot blot is used to detect Antidiacyltrehalose [[Antibodies]] in [[Tuberculous]] patients and [[Typhoid Fever]]. This test could increase the number of lives saved that are affected with these diseases. Using the dot blot test can be useful for under-developed countries especially. The dot blot is a good positive predictor of these diseases for countries and regions lacking in medical facilities and laboratories. By using this test many lives could be saved and a cure could be found for the types of diseases this test can detect<!--{{Fact}} begin-->{{fix
|link=Wikipedia:Citation needed
|text=citation needed
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== See also ==
* [[Southern blotELISpot]]
* [[Northern blot]]
* [[Western blot]]
* [[Southwestern blot]]
 
== External linksReferences ==
{{reflist}}
*[http://www.bio-rad.com/cmc_upload/Literature/12484/M1706542C.pdf A protocol for the BioRad Dot-Blot Apparatus]
 
[[Category:MolecularGenetics biologytechniques]]▼
== Citations and More Information ==
[[Category:Molecular biology techniques]]
*[http://cvi.asm.org/content/7/2/312.full]
*[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95755/]
*[http://www.abcam.com/ps/pdf/protocols/Dot%20blot%20protocol.pdf]
*[http://www.motherlandinc.org/clinic/index.php?option=com_content&task=view&id=16&Itemid=9]
*[http://www.biosynth.com/index.asp?topic_id=144]
▲[[Category:Molecular biology]]
 
 
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