ashwini blotting tech seminar

BLOTTING TECHNIQUES

Presented by: Ashwini PatilM. Pharm (Pharmacology) Sem: IIDepartment of PharmacologyR.C Patel Institute of Pharmaceutical Education and Research, Shirpur

HISTORY OF BLOTTING

Southern blotting is named after its inventor, the British biologist Edwin Southern (1975)

Other blotting methods Western (WB), Northern (NB) that employs similar principles, but using probes or RNA, have later been named in reference to Edwin Southern name

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Blotting Visualisation of specific DNA ,RNA & Protein

among many thousands of contaminating molecules requires no. of techniques which are collectively termed BLOT transfer and the process is called as Blotting.

A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier

(for example, a nitrocellulose, polyvinylidene fluoride (PVDF) or nylon membrane)

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TYPES OF BLOTTING TECHNIQUES

Southern blotting

Western blotting

Northern blotting

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1)Far western blot2)Far eastern blot3)Reverse northern blot4)Dot blot

Southern Blotting PRINCIPLE:- The key to this method is Hybridization. Hybridization:-It is a process of forming a double

stranded DNA molecule between a single stranded DNA probe and single stranded target DNA.

There are two important features of Hybridization i.e

1)The reactions are specific-the probes will bind to targets with complementary sequence. 2)The probe can find one molecule of target in a mixture of millions of related but non complementary molecules.

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Procedure5

Technique and Apparatus 6

Interpretation of Technique 7

Applications To identify specific DNA in a DNA sample. To isolate desired DNA for construction of DNA. To identify mutations, deletions, and gene

rearrangements. Used in prognosis of cancer and in prenatal

diagnosis of genetic disease. Used in phylogenetic analysis. Diagnosis of HIV-1 and infectious disease. In DNA fingerprinting1) Paternity and maternity testing2) Criminal identification and forensics3)Personal identification

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Northern blotting

Northern blotting is a technique for detection of specific RNA sequences. Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting.

PRINCIPLE:-1)Hybridization 2) Electrophoresis3) Capillary action

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ProcedureExtract and purify mRNA from cells.

Separated by gel electrophoresis

Transfer to aminobenzyloxymethyl filter paper.(BLOTTING)

Add labelled DNA probe for hybridization to take place

Wash off unbound probe

autoradiograph to detect mRNA- DNA hybrid.

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Detecting a specific mRNA in a sample. Used in the screening of recombinants by detecting

the mRNA produced by transgenesis. In disease diagnosis. In gene expression studies. mRNA splicing studies. Study RNA degradation. Study RNA half life.

Applications

Disadvantages Time consuming procedure. RNA samples can be degraded by RNAases. Use of radioactive probes. Detection of multiple probe is a problem.

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The reverse northern blot is a method by which gene expression patterns may be analyzed by comparing isolated RNA molecules from a tester sample to samples in a control cDNA library.

Reverse northern blot 13

Applications Quantification of mRNA expression levels Confirmation of differential display results DNA Microarrays.

Research Applications:-1)Reverse northern blotting was used in a 2013 study in Gene in which the author identified a number of genes responsible for early cold-resistance response in the cold-hardy citrus fruit Poncirus trifoliata.

2)A study utilized the technique to determine differences in striatal tissue in rats treated with 3-NP, which is often used in experiments to generate a Huntington’s Disease-like phenotype in rats.

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Procedure

1• cDNA sequences for transcripts of interest are

immobilized on nylon membranes

2• Prepared reverse northern blot membranes are

pre-hybridized in Denhardt’s solution with SSC buffer and labeled cDNA probes are denatured at 100 °C and added to the pre-hybridization solution.

3• The membrane is incubated with the probes for

at least 15 hours at 65 °C, then washed and exposed.[3]

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Western blotting Western blotting uses specific antibodies to identify

proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride).

The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane.

The membrane can then be further processed with antibodies specific for the target of interest, and visualized using secondary antibodies and detection reagents.

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Principle:- It is based on the principle of immunochromatography.

Antigens are separated by Poly Acrylomide Gel Electrophoresis(PAGE).

Antibodies in serum react with specific antigens. Signals are detected according to the principles of test

systems.

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Procedure The technique

uses three elements

(1) separation by size (2) Transfer to a solid support (3) Marking target protein using a proper primary and secondary antibody to visualize.

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Apparatus

 Thermo Scientific myECL Imager

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Detection 21

Advantages Used to pinpoint a specific protein in a given

sample, employs the ability of an enzyme or fluorescence-labeled primary antibody to bind to its specific antigen.

Sensitivity:-Its ability to detect as little as 0.1 nanograms of protein in a sample.

Fewer antibodies are needed for testing, which cuts down laboratory costs significantly.

Specificity:-specific antibodies show affinity for specific proteins, the process can selectively detect a target protein even in a mixture of 3 lakh different proteins.

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Prone to False or Subjective Results:- A false-positive results comes when an antibody reacts with a non-intended protein, which is what frequently happens when a patient being tested for HIV.

On the other hand false result can easily occur if larger proteins are not given sufficient time to transfer properly to the membrane.

High Cost and Technical Demand:-A delicate process, western blotting requires precision in every step for proper identification of a sample's constituents.

Disadvantages 23

Highly sensitive method to detect a specific protein even in very low quantity.

Used in clinical diagnosis Quantifying a gene product (

gene expression studies) Some forms of Lyme disease testing

employ Western blotting.

Applications 24

Far western blot

To identify proteins that interact with a given protein.Far-Western blot analysis is an alternative method to analyze protein-protein interactions.

PRINCIPLE:-The principle of far-western blotting is modified from western blotting.

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Procedure

1• Uses the natively-folded recombinant protein

as the first probe to interact with proteins that have been separated on a PVDF membrane.

2• The second probe used in this platform is the

specific antibody against the original protein, while the HRP-conjugated third probe is the antibody against the second probe.

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• When combined with protein identification using LC-MS-MS, the resulting protein spots (or protein bands) can be characterized and then subjected to further functional assays, such as protein immuno-precipitation.

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Advantages Low experimental cost. High sensitivity.

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Far eastern blotting

Far-Eastern blotting is a technique developed in 1994 by Taki and colleagues

at the Tokyo Medical and Dental University, Japan for the analysis of lipids separated by high-performance thin layer chromatography.

The lipids are transferred from the HPTLC plate to a PVDF membrane for further analysis, for example by enzymatic or ligand binding assays or mass spectrometry.

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Procedure 29

Applications

Purification of phospholipids. Structural analysis of lipids in conjunction

with direct mass spectrometry. Binding study using various ligands such

as antibodies, lectins, bacterium, viruses, and toxins.

Enzyme reaction on membranes.

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Dot blot method

A technique for detecting, analyzing and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate.

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Procedure 32

Application

Estimation of concentration of proteins in crude preparations (such as culture supernatant).

The size of complementary sequences is obtained through the process.

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References

http://ourpastimes.com/advantages-disadvantages-western-blot-8670663.html

http://www.biologyexams4u.com/2014/01/western-blotting-principle-summary-of.html

https://www.researchgate.net/figure/231212350_fig1_Figure-1-Schematic-diagram-of-immunochromatography

http://www.biologyexams4u.com/2013/12/southern-blotting-procedure-steps.html

http://i-base.info/guides/testing/test-accuracy-results-and-further-testing

http://www.aidsmap.com/Confirmatory-tests/page/1323369/

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http://labtestsonline.org.uk/understanding/analytes/lyme/tab/test/

http://www.drzeegers.com/2014/08/lyme-disease-diagnosis-blood-test-tickborne-disease/

http://www.biologyexams4u.com/2014/01/northern-blotting-principle-summary-of.html

Sritunyalucksana K, Wannapapho W, Lo CF, Flegel TW. (2006) PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp. J Virol 80: 10734-10742

www.scribd.com http://webap.rsh.ncku.edu.tw/shrimpw

ssv/index.php/sfgc-technological-platforms/protein-interactions-using-far-western-blotting

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http://www.wikivisually.com/wiki/Reverse_Northern_blot

http://www.abcam.com/protocols/dot-blot-protocol

http://feedforbiotech.blogspot.com/2011/03/dot-blot.html

https://www.thermofisher.com (far western)

https://fr.wikipedia.org/wiki/Far-eastern_blot

https://www.pathwayz.org/Tree/Plain/TRANSGENESIS

https://openi.nlm.nih.gov(mrna degradation)

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http://www.tiem.utk.edu/~gross/bioed/webmodules/mRNA.htm

http://bitesizebio.com/7206/introduction-to-dna-microarrays/

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