Transcript of "Southern northern and western blotting"
Comparison of Southern,Northern, and Western analysesof Gene X
Southern hybridization First described by E. M. Southern in 1975. Applications of Southern hybridization RFLP’s, VNTR’s and DNA fingerprinting Checking of the gene knockout mice The flow chart of Southern hybridization
Flow chart of Southernhybridization Preparing the samples and running the gel Southern transfer Probe preparation Isotope Non-isotope Prehybridization Hybridization Post-hybridization washing Signal detection
Preparing the samples andrunning the gel Digest 10 pg to 10 µg of desired DNA samples to completion. Prepare an agarose gel, load samples (remember marker), and electrophorese. Stain gel ethidium bromide solution (0.5 µg/ml). Photograph gel (with ruler).
Critical parameters (I) Note the complexity of DNA Genomic DNA A single-copy of mammalian gene, 3 Kb average in length 10 µg x 3 Kb/3 x 106 Kb = 10 µg x 1/106 = 10 pg Plasmid DNA or PCR products 0.1 µg of a 3 Kb plasmid DNA ≅100 ng
Gel treatment Acid treatment 0.2 N HCl solution Denaturation NaOH solution Neutralization Tris-Cl buffer (pH8.0)
Southern transfer Measure gel and set up transfer assembly: Wick in tray with 20x SSC Gel Nitrocellulose or Nylon filters (soaked in H2O and 20x SSC) 3MM Whatman filter paper Paper towels Weight
After Southern transfer Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC Bake nitrocellulose at 80°C for 2 hr or UV-crosslink Nylon membrane for seconds
Preparation of probes Synthesis of uniformly labeled double-stranded DNA probes Preparation of single-stranded probes Labeling the 5′ and 3′ termini of DNA
Synthesis of double-stranded DNAprobes - Nick translation of DNA - Labeled DNA probes using random oligonucleotide primers
Preparation of single-strandedprobes Synthesis of single-stranded DNA probes using bacteriophage M13 vectors. Synthesis of RNA probes by in vitro transcription by bacteriophage DNA- dependent RNA polymerase.
Labeling the 5′ and 3′ termini of DNA Labeling the 3′ termini of double-stranded DNA using the Klenow fragment of E. coli DNA polymerase I. (lack of 5’ 3’ exonuclease activity) Labeling the 3′ termini of double-stranded DNA using bacteriophage T4 DNA polymerase. Labeling the 5′ termini of DNA with bacteriophage T4 polynucleotide kinase.
PCR Labeling, Random PrimedLabeling, and RNA Labeling
Prehybridization Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr
Hybridization Remove prehybridization solution and add hybridization solution Add 500,000 cpm of the probe/ml hybridization solution. Hybridize overnight at appropriate temperature.
Post-hybridization washing Wash twice, 15 min each, in 1x SSC, 0.1% SDS at room temperature. Wash twice, 15 min each, in 0.25x SSC, 0.1%SDS at hybridization temp
Critical parameters (II) Homology between the probe and the sequences being detected Tm = 81 +16.6 (log Ci) + 0.4 [% (G+C)] - 0.6 (% formamide)- 600/n - 1.5 (% mismatch) Factors can be changed: Hybridization temp. Washing temp. Salt concentration during washing High temp., low salt: high stringency Low temp., high salt: low stringency If 50 % formamide is used 42 oC for 95 ~ 100 % homology 37 oC for 90 ~ 95 % homology 32 oC for 85 ~ 90 % homology
Comparison of nitrocelluloseand nylon membranes NC Nylon Hydrophobic binding Covalent binding Fragile Durable Probe length > 200 ~ < 200 ~ 300 bp is 300 bp O.K. Lower background Higher background Cannot be exposed Can be exposed to to basic solution basic solution Not easily Can be reprobed reprobed several times
Northern blotting or Northernhybridization Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe.
The flow chart of Northernhybridization samples and run RNA gel Prepare RNA Northern transfer Probe preparation Isotope Prehybridization Non-isotope Hybridization Post-hybridization washing Signal detection
Preparation ofagarose/formaldehyde gel E.g. Prepare a 350 ml 1.2% agarose/formaldehyde gel 4.2 g agarose in 304.5 g water. Microwave, then cool to 60°C. Add 35 ml 10x MOPS running buffer and 10.5 ml 37% formaldehyde
Preparation of RNA samples Prepare a premix: 5 µl of 10x MOPS running buffer 8.75 µl of 37% formaldehyde 25 µl of formamide. Prepare RNA samples: 38.75 µl of premix RNA (0.5 to 10 µg)* water to 50 µl *If the mRNA species of interest makes up a relatively high percentage of the mRNA in the cell (>0.05% of the message), total cellular RNA can be used. If the mRNA species of interest is relatively rare, however, it is advisable to use poly(A)+ RNA. Incubate 15 min at 55°C
Running the RNA gel Add 10 µl formaldehyde loading buffer to each sample and load gel. Run gel at 100 to 120 V for ~3hr. Remove gel from the running tank and rinse several times in water. Place gel in 10x SSC for 45 min. Do not need post-transferring gel treatment
An example of Northern blottingNorthern blot RNA gel 28 S 18 S
Western blotting, orimmunoblottingTechnique for detecting specific proteinsseparated by electrophoresis by use oflabeled antibodies.
Flow chart of Western blotting Electrophoresing the protein sample Assembling the Western blot sandwich Transferring proteins from gel to nitrocellulose paper Staining of transferred proteins Blocking nonspecific antibody sites on the nitrocellulose paper Probing electroblotted proteins with primary antibody Washing away nonspecifically bound primary antibodyDetecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitate Photographing the immunoblot
Analysis of protein samples by SDSpolyacrylamide-gel electrophoresis andWestern blotting Protein bands detected by specific antibody SDS-PAGE Western blot
Comparison of Southern, Northern,and Western blotting techniques Southern blotting Northern blotting Western blotting Molecule DNA (ds) mRNA (ss) Protein detected Gel Agarose gel Formaldehyde Polyacrylamide gelelectrophoresis agarose gel Gel Depurination, - - pretreatment denaturation, and neutralizationBlotting method Capillary transfer Capillary transfer Electric transfer Probes DNA cDNA, cRNA primary antibody Radioactive or Radioactive or nonradioactive nonradioactive Detection Autoradiography Autoradiography Chemiluminescent system Chemiluminescent Chemiluminescent Colorimetric Colorimetric Colorimetric
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