INTRODUCTION: In molecular biology and genetics, a blot is a method of transferring proteins, DNA or RNA, onto a carrier (for example,

a nitrocellulose PVDF or nylon membrane ! 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 "isuali#ed by one or more different methods$

colorant staining (for example, sil"er staining of proteins autoradiographic "isuali#ation of radioacti"e labelled molecules (performed before the blot specific labelling of some proteins or nucleic acids! It is done %ith antibodies or hybridi#ation probes that bind only to some molecules of the blot and ha"e an en#yme &oined to them! After proper %ashing, this en#ymatic acti"ity (and so, the molecules %e search in the blot is "isuali#ed by incubation %ith proper reacti"e, rendering either a colored deposit on the blot or a chemiluminiscent reaction %hich is registered by photographic film!

'ommon blot methods are $

(outhern blot for DNA (outh%estern blot for Protein)DNA Northern blot for RNA Re"erse Northern blot for RNA *estern blot for proteins Far)*estern blot for Protein)Protein +astern blotting for posttranslational modification Far)+astern blot for ,ipids, Drugs and -ormones Dot blot (lot blot

SOUTHERN BLOT: A Southern blot is a method routinely used in molecular biology for detection of a specific DNA se.uence in DNA samples! (outhern blotting combines transfer of electrophoresis)separated DNA fragments to a filter membrane and subse.uent fragment detection by probe hybridi#ation! /he method is named after its in"entor, the 0ritish biologist +d%in (outhern!

1ther blotting methods (i!e!, *estern blot,234 Northern blot, +astern blot, (outh%estern blot that employ similar principles, but using RNA or protein, ha"e later been named in reference to +d%in (outhern5s name! As the techni.ue %as eponymously named, (outhern blot is capitali#ed as is con"entional for proper nouns! /he names for other blotting methods may follo% this con"ention, by analogy! Method
6! Restriction endonucleases are used to cut high)molecular)%eight 3! 7!

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DNA strands into smaller fragments! /he DNA fragments are then electrophoresed on an agarose gel to separate them by si#e! If some of the DNA fragments are larger than 68 9b, then prior to blotting, the gel may be treated %ith an acid, such as dilute -'l, %hich depurinates the DNA fragments, brea9ing the DNA into smaller pieces, thus allo%ing more efficient transfer from the gel to membrane! If al9aline transfer methods are used, the DNA gel is placed into an al9aline solution (typically containing sodium hydroxide to denature the double)stranded DNA! /he denaturation in an al9aline en"ironment may impro"e binding of the negati"ely charged DNA to a positi"ely charged membrane, separating it into single DNA strands for later hybridi#ation to the probe (see belo% , and destroys any residual RNA that may still be present in the DNA! /he choice of al9aline o"er neutral transfer methods, ho%e"er, is often empirical and may result in e.ui"alent results!2citation needed4 A sheet of nitrocellulose (or, alternati"ely, nylon membrane is placed on top of (or belo%, depending on the direction of the transfer the gel! Pressure is applied e"enly to the gel (either using suction, or by placing a stac9 of paper to%els and a %eight on top of the membrane and gel , to ensure good and e"en contact bet%een gel and membrane! If transferring by suction 3;< ((' buffer is used to ensure a seal and pre"ent drying of the gel! 0uffer transfer by capillary action from a region of high %ater potential to a region of lo% %ater potential (usually filter paper and paper tissues is then used to mo"e the DNA from the gel on to the membrane= ion exchange interactions bind the DNA to the membrane due to the negati"e charge of the DNA and positi"e charge of the membrane!

>! /he membrane is then ba9ed in a "acuum or regular o"en at ?; @' for

3 hours (standard conditions= nitrocellulose or nylon membrane or exposed to ultra"iolet radiation (nylon membrane to permanently attach the transferred DNA to the membrane! A! /he membrane is then exposed to a hybridi#ation probeBa single DNA fragment %ith a specific se.uence %hose presence in the target DNA is to be determined! /he probe DNA is labelled so that it can be detected, usually by incorporating radioacti"ity or tagging the molecule %ith a fluorescent or chromogenic dye! In some cases, the hybridi#ation probe may be made from RNA, rather than DNA! /o ensure the specificity of the binding of the probe to the sample DNA, most common hybridi#ation methods use salmon or herring sperm DNA for bloc9ing of the membrane surface and target DNA, deioni#ed formamide, and detergents such as (D( to reduce non) specific binding of the probe! ?! After hybridi#ation, excess probe is %ashed from the membrane (typically using ((' buffer , and the pattern of hybridi#ation is "isuali#ed on <)ray film by autoradiography in the case of a radioacti"e or fluorescent probe, or by de"elopment of color on the membrane if a chromogenic detection method is used! NORTHERN BLOT: /he northern blot is a techni.ue used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA in a sample! Procedure A general blotting procedure starts %ith extraction of total RNA from a homogeni#ed tissue sample! /he mRNA can then be isolated through the use of oligo (d/ cellulose chromatography to maintain only those RNAs %ith a poly(A tail! RNA samples are then separated by gel electrophoresis! (ince the gels are fragile and the probes are unable to enter the matrix, the RNA samples, no% separated by si#e, are transferred to a nylon membrane through a capillary or "acuum blotting system!

'apillary blotting system setup for the transfer of RNA from an electrophoresis gel to a blotting membrane!

A nylon membrane %ith a positi"e charge is the most effecti"e for use in northern blotting since the negati"ely charged nucleic acids ha"e a high affinity for them! /he transfer buffer used for the blotting usually contains formamide because it lo%ers the annealing temperature of the probe)RNA interaction pre"enting RNA degradation by high temperatures! 1nce the RNA has been transferred to the membrane it is immobili#ed through co"alent lin9age to the membrane by CV light or heat! After a probe has been labeled, it is hybridi#ed to the RNA on the membrane! +xperimental conditions that can affect the efficiency and specificity of hybridi#ation include ionic strength, "iscosity, duplex length, mismatched base pairs, and base composition!/he membrane is %ashed to ensure that the probe has bound specifically and to a"oid bac9ground signals from arising! /he hybrid signals are then detected by <)ray film and can be .uantified by densitometry! /o create controls for comparison in a northern blot, samples not displaying the gene product of interest can be used after determination by microarrays or R/)P'R! Gels RNA run on a formaldehyde agarose gel to highlight the 3?( (top band and 6?( (lo%er band ribosomal subunits! /he RNA samples are most commonly separated on agarose gels containing formaldehyde as a denaturing agent for the RNA to limit secondary structure! /he gels can be stained %ith ethidium bromide (+t0r and "ie%ed under CV light to obser"e the .uality and .uantity of RNA before blotting! Polyacrylamide gel electrophoeresis %ith urea can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs!2634 An RNA ladder is often run alongside the samples on an electrophoresis gel to obser"e the si#e of fragments obtained but in total RNA samples the ribosomal subunits can act as si#e mar9ers! (ince the large ribosomal subunit is 3?( (approximately 89b and the small ribosomal subunit is 6?( (approximately 39b t%o prominent bands %ill appear on the gel, the larger at close to t%ice the intensity of the smaller!