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Macromolecule Blotting

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Macromolecule Blotting 1 Macromolecule Blotting Description of Module Subject Name Paper Name Module Name/Title Macromolecule Blotting Dr. Vijaya Khader Dr. MC Varadaraj 2 Macromolecule Blotting 1. Objectives 1. Blotting/Hybridization and its principle 2. Types of Blotting 3. Applications 2. Lay Out 3 Macromolecule Blotting Blotting Southern Northern Western 4 Macromolecule Blotting 3. 1 Description Blotting means to transfer DNA or RNA to nitrocellulose membrane and probe binds to complementary strand in the sample to give result in the form of signal using radioactive or biotin labeled probe. Principle At high temperature two strands of DNA get separated they and re-anneal when temperature is brought back to normal. There are 2 important features of hybridization: The reaction is very specific that means the probes will only bind to targets with a complementary sequence. The probe can identify one molecule of target in a mixture of millions of related and non- complementary molecules 2 Types of Blotting 2.1 Southern Blot: It is a method in molecular biology which is used for the identification of a specific DNA sequence in a mixture of DNA samples. In this DNA is first run on agarose gel and than transferred to membrane and further detection using specific probe. This method is known by its inventor’s name who was biologist by profession, Edwin Southern. After this other methods of blotting are named Northern and western further in continuation to Southern blot. 2.2 Northern Blot: The northern blot is technique used in molecular biology research to study expression of by detecting RNA (or isolated mRNA) in a sample. Northern blotting involves the use of agarose gel electrophoresis to separate RNA samples by size, and detects with a hybridization probe which is complementary target sequence. Blot means to transfer RNA from the electrophoresis gel to the blotting membrane. This technique was given in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. 5 Macromolecule Blotting 2.3 Western Blot: The western blot is a widely used analytical technique used to detect specific proteins in a sample. It uses SDS to separate proteins. The proteins from the SDS gel are then transferred to a nitrocellulose or PVDF membrane, and incubated with antibodies specific to the target protein. 2.4 Eastern Blot: To study post translational modification this technique is (lipids, phosphomoieties and glycoconjugates). It is mostly used to detect carbohydrate epitopes. Eastern blotting is considered an extension of the Western blotting. Transferred proteins are analyzed for post- translational modifications using specific probes that detect lipids, carbohydrate, phosphorylation or any other protein modification. 3 Probe: Is defined sequence which is used to search mixtures of nucleic acids for molecules containing a complementary sequence. Any positive sample can be used as control (eg. In diagnosis of viruses) Blotting Membranes Membrane or support onto which the separated proteins are transferred which bind proteins with high affinity: • Nitrocellulose membrane • has very good protein binding capacity and retention ability • Polyvinylidene fluoride (PVDF) membrane • Its strength is more than nitrocellose membrane so it can be used if reprobing and stripping is required Summary of steps Run samples on a gel/make a slot/dot blot Transfer to a membrane Link sample to membrane 6 Macromolecule Blotting Make a probe Hybridize probe to membrane Detect probe Labeling of DNA or RNA probes Radioactive labeling: Use of radioactive material for labelling Non-radioactive labeling: Enzymatic labeling using biotin labeled probes End labeling: Labels at the ends Uniform labeling: labels internally Hexanucleotide primered labeling: After denaturing DNA add random hexamer primers and DNA polymerase Known viral gene sequence (or any other DNA/RNA ) run in 1% agarose gel. Viral DNA fragment is eluted from the gel and incubated in boiling water bath for 10 min, for denaturation of DNA Table: Probe preparation mix Denatured DNA 200-500 ng Random Primer 100 ng 10x Klenow buffer 3 µl dNTP mix (-CTP)(3.3nM each) 4.5 µl α-32P dCTP (10µCi/µl,specific activity 3x103 Ci/mmole) 10 µCurie Klenow enzyme 5 units 7 Macromolecule Blotting Final volume with ddH2O 30 µl The reaction is incubated at 37°C for one hour. To this, equal volume of Buffer-A is added. The mixture is then denatured by incubating the tube in boiling water bath for 7 min. The contents are immediately transferred to ice before adding to the hybridization bottle. dNTP mix: (for α32-P dCTP as the radioactive molecule, 100mM stock of dATP, dTTP and dGTP): 1+1+1+27µl water. 4.5µl of this mix is used for one reaction. Buffer-A: 500mM Tris HCL (pH 7.5), 500mM NaCl, 5mM EDTA and 0.5% SDS. Blotting in virus diagnostics(Southern and Northern) Procedure for northern and Southern blot is same except that in Southern DNA is used and in Northern RNA is used. Probes prepared from cloned viral cDNAs are used in Southern blots to detect the viral DNA in infected plants. A modification in this technique called tissue printing employs blotting of leaf squashes rather than the viral DNA on to nylon membranes . Use of radioactive material for preparing the probes has been the major limitation of this technique. To avoid this hazardous material, non-radioactive labelling techniques have been developed 6.1 Diagnosis of RNA and DNA viruses Slot-Blot Hybridization Samples are crushed in DNA/RNA isolation buffer and centrifuged to remove debris (CTAB Buffer or TRI reagent etc.) 8 Macromolecule Blotting Approx 200µl liquid sample is denatured in a boiling water bath for 5 min before loading. The slot-blot manifold is assembled with positively charged nylon membrane, previously wetted with sterile water. 9 Macromolecule Blotting Slot -blot and dot-blot apparatus 10 Macromolecule Blotting Figure: Membrane after dot-blot transfer Procedure The manifold is connected to vacuum pump. To each well, 10x SSC buffer (200µl) is added and vacuum was applied till the buffer is completely absorbed but not dried. After that, 200µl each of the denatured samples is loaded to the wells Vacuum is applied till the samples is absorbed completely. Afterwards, 200µl of 10x SSC is added and allowed to completely pass through. 20x SSC Buffer: 3M NaCl and 0.3 M Trisodium citrate Vacuum is released, apparatus disassembled and the membrane is rinsed in 2x SSC. The membrane is air dried and exposed to UV light for 2 min in a UV crosslinker for binding of the transferred nucleic acids. The membrane is wrapped in saran film and stored at 4°C until hybridization. After completion of electrophoresis, the DNA in the gel is denatured by placing the gel in a plastic tray containing the denaturation solution with slow shaking on a gel rocker for 30 min. After denaturation, the gel is soaked in neutralizing solution for 30 min with slow shaking. The two chambers of the electrophoresis tank are filled with transfer solution (10x SSC). A wick of Whatman® 3MM paper is soaked in 10x SSC and placed over the gel platform of the tank with its ends submerged in the solution. 11 Macromolecule Blotting Denaturing Solution: 1M NaCl and 0.5M NaOH Neutralizing Solution: 1.5M Tris HCl (pH 8) and 3M NaCl Three more sheets of Whatman® 3MM paper (slightly bigger than the gel) are soaked in 10x SSC and placed over the wick. The gel is carefully inverted and placed over the sheets avoiding any air bubbles between the gel and Whatman® papers. Nylon membrane is cut to the gel size, wetted in sterile water and placed over the gel avoiding any air bubbles. Three more sheets of Whatman® 3MM paper are cut to gel size, soaked in 10x SSC and placed over the membrane. A stack of gel-sized dry blotting papers (8-10 cm height) are placed over the 3MM papers and approximately 500 g weight is placed on top of the stack. The transfer is allowed to take place for 14-16 h. 12 Macromolecule Blotting Figure: Capillary Transfer of DNA from gel to the membrane After completion of the transfer, the blotting paper stack is removed, the membrane is marked so as to indicate the orientation and rinsed in 2x SSC briefly. The buffer is removed by placing the membrane between the folds of a dry 3MM sheet but the membrane is not completely dried. 13 Macromolecule Blotting The membrane is wrapped in a cling film and placed under UV light in a UV cross-linker for 2 min for cross-linking the DNA to the membrane. The blot is then stored at 4C until hybridization. Figure: Hybridization bottle and oven Prehybridization Pre-hybridization is carried out for 1 hr at 42°C in the pre-hybridization buffer. Pre-hybridization buffer consists of: Formamide 50% (v/v) Na2HPO4 pH 7.2 120 mM NaCl 250 mM EDTA, pH 8.0 1 mM SDS (w/v) 7% After pre-hybridization, probe is added to fresh pre-hybridization solution and incubated at 42°C overnight (10-18 hrs) and this step is called hybridization Washing of Blots 14 Macromolecule Blotting After hybridization is complete the blots are removed from the hybridization solution, rinsed briefly in 2x SSC and placed in the first washing buffer followed by 2nd and third wash with progressive stringency as follows: First Wash Buffer 2.0x SSC, 0.1% SDS (25°C) - two washings of 15 min each Second wash buffer 0.5x SSC, 0.1% SDS (25°C) - two washings of 15 min each Third Wash buffer 0.1x SSC 0.1% SDS (65°C) - two washings of 30 min each After washing, the blots are placed on a Whatman filter paper to remove excess of liquid and wrapped in a saran wrap immediately, to prevent it from drying and for the purpose of autoradiography.
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