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Southern, Northern and Western blotting. Comparison of Southern, Northern, and Western analyses of Gene X.

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Presentation on theme: "Southern, Northern and Western blotting. Comparison of Southern, Northern, and Western analyses of Gene X."— Presentation transcript:

1 Southern, Northern and Western blotting

2 Comparison of Southern, Northern, and Western analyses of Gene X

3 Southern hybridization First described by E. M. Southern in Applications of Southern hybridization RFLP s, VNTR s and DNA fingerprinting Checking of the gene knockout mice The flow chart of Southern hybridization

4 Southern hybridization Transfer buffer

5 Detection of an RFLP by Southern blotting

6 Detection of the sickle-cell globin gene by Southern blotting

7 Checking of the gene knockout mice

8 Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection Isotope Non-isotope

9 Preparing the samples and running the gel Digest 10 pg to 10 g of desired DNA samples to completion. Prepare an agarose gel, load samples (remember marker), and electrophorese. Stain gel ethidium bromide solution (0.5 g/ml). Photograph gel (with ruler).

10 Critical parameters (I) Note the complexity of DNA Genomic DNA A single-copy of mammalian gene, 3 Kb average in length 10 g x 3 Kb/3 x 10 6 Kb = 10 g x 1/10 6 = 10 pg Plasmid DNA or PCR products 0.1 g of a 3 Kb plasmid DNA 100 ng

11 Gel treatment Acid treatment 0.2 N HCl solution Denaturation NaOH solution Neutralization Tris-Cl buffer (pH8.0)

12 Southern transfer Measure gel and set up transfer assembly: Wick in tray with 20x SSC Gel Nitrocellulose or Nylon filters (soaked in H 2 O and 20x SSC) 3MM Whatman filter paper Paper towels Weight

13 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

14 Preparation of probes Synthesis of uniformly labeled double-stranded DNA probes Preparation of single-stranded probes Labeling the 5 and 3 termini of DNA

15 Synthesis of double-stranded DNA probes - Nick translation of DNA - Labeled DNA probes using random oligonucleotide primers

16 Nick translation

17 Preparation of single-stranded probes Synthesis of single-stranded DNA probes using bacteriophage M13 vectors. Synthesis of RNA probes by in vitro transcription by bacteriophage DNA- dependent RNA polymerase.

18 In vitro transcription

19 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. Labeling the 5 and 3 termini of DNA

20 T4 polynucleotide kinase activity

21 Non-isotope labeling Digoxigenin-11-dUTP (DIG-dUTP) labeling - DNA labeling - Oligonucleotide labeling - RNA labeling

22 PCR Labeling, Random Primed Labeling, and RNA Labeling

23 Prehybridization Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr

24 Hybridization Remove prehybridization solution and add hybridization solution Add 500,000 cpm of the probe/ml hybridization solution. Hybridize overnight at appropriate temperature.

25 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

26 Critical parameters (II) Homology between the probe and the sequences being detected Tm = (log Ci) [% (G+C)] (% formamide)- 600/n (% 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 o C for 95 ~ 100 % homology 37 o C for 90 ~ 95 % homology 32 o C for 85 ~ 90 % homology

27 Comparison of nitrocellulose and nylon membranes NCNylon Hydrophobic bindingCovalent binding FragileDurable Probe length > 200 ~ 300 bp < 200 ~ 300 bp is O.K. Lower backgroundHigher background Cannot be exposed to basic solution Can be exposed to basic solution Not easily reprobed Can be reprobed several times

28 Signals detection Autoradioragraphy Non-isotope detection system - Chemiluminescent detection - Colorimetric detection - Multicolor detection

29 Autoradiography Exposure to x-ray film

30 Northern blotting or Northern hybridization Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe.

31 The flow chart of Northern hybridization Prepare RNA samples and run RNA gel Northern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection Isotope Non-isotope

32 Preparation of agarose/formaldehyde gel E.g. Prepare a 350 ml 1.2% agarose/formaldehyde gel 4.2 g agarose in g water. Microwave, then cool to 60 C. Add 35 ml 10x MOPS running buffer and 10.5 ml 37% formaldehyde

33 Preparation of RNA samples Prepare a premix: 5 l of 10x MOPS running buffer 8.75 l of 37% formaldehyde 25 l of formamide. Prepare RNA samples: l of premix RNA (0.5 to 10 g)* water to 50 l *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

34 Running the RNA gel Add 10 l 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

35 An example of Northern blotting Northern blot RNA gel 28 S 18 S

36 Western blotting, or immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies.

37 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 antibody Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitate Photographing the immunoblot

38 SDS polyacrylamide-gel electrophoresis (SDS-PAGE)

39 Analysis of protein samples by SDS polyacrylamide- gel electrophoresis and Western blotting Protein bands detected by specific antibody SDS-PAGE Western blot

40 Comparison of Southern, Northern, and Western blotting techniques


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