Blotting technique

It is speak about Blotting technique, Western,Northern,Southern blot.It is collect from different power point and source.

1. Blotting technique Collect and summarized by Murtadha Ali AL- Khegane M.S.C student

2. Definition and types is a technique by which a macromolecule such as DNA, RNA, or protein is resolved in a gel matrix, transferred to a solid support, and detected with a specific probe Visualization of specific DNA , RNA & protein among many thousands of contaminating molecules. Types : 1 ) Southern blotting ( to detect DNA ) 2 ) Northern blotting ( to detect RNA ) 3 ) Western blotting ( to detect protein )

3. Southern blotting (DNA) invented by the English Molecular Biologist Edwin Southern (1975) Steps: DNA Fragmentation Agarose Gel Electrophoresis Depurination (optional) Neutralization Blotting Prehybridization and Hybridization Removal of Unbound Probe Autoradiograph

4. 1. DNA Fragmentation: DNA is digested by one or several restriction enzymes ( bacterial enzymes ) - - cut at specific sequence (restriction site) 2. Agarose Gel Electrophoresis: Restriction fragments are separated electrophoretically by size on agarose gel . DNA fragments migrate into gel toward the anode (+ ve electrode) under the influence of electric field because it is negatively charged

5. 3. De- purination (Optional): When DNA fragments is > 15 kilobases , it is too hard to be transferred to filter The gel is treated with dilute acid (0.2 M HCl for 15 minutes) to have smaller pieces can trans. 4. Neutralization: DNA is placed into an alkaline solution containing 0.5mM NaOH to denature the dsDNA into ssDNA and neutralize the acid in previous step. Function of Neutralization : (1) improve binding of the – ve charged DNA to + ve charged filter (2) ssDNA strands for hybridization (3) destroy any remaining RNA present in the sample

6. 5. Blotting: I-Cover gel with nitrocellulose paper. II-Cover nitrocellulose paper with thick layer of paper towels. III-Compress apparatus with heavy weight. IV- ssDNA binds to nitrocellulose at same position it had on the gel. V- Vacum dry nitrocellulose at 80  C to permanently fix DNA in place or cross link (via covalent bonds) the DNA to the membrane.

7. 6 . Hybridization: Incubate nitrocellulose sheet with a minimal quantity of solution containing 32 P-labeled ssDNA probe. Probe sequence is complementary to the DNA of interest. 7. Removal of Unbound Probe : Unbound probe is washed off.

8. 8.Autoradiograph: is an image on an  x-ray   film The location of the probe is revealed by converting a colorless substrate to a colored product that can be seen or gives off light which will expose X-ray film. The bands indicate the number and size of the DNA fragments complementary to the probe.

9. Comparison of Nitrocellulose, Nylon membranes Nitrocellulose membrane Nylon membrane DNA- binding capacity 100 µg/cm 500 µg/cm Stability fragile less fragile than nitrocellulose filter Remarks Non-specific binding site is easily blocked Protein staining is difficult owing to the + ve charged membrane.

10. Digest DNA with restriction enzymes 1 Blotting. Transfer separated DNA onto membrane for further analysis 3 Hybridize the target DNA with specific labeled probe 5 Restriction Fragments are separated by size by agarose gel 2 Synthesis of labelled probe 4 Wash the filter and expose the film to x-ray 6

11. Main functions Detect the specific DNA sequence (gene) of interest Determine the length of the restriction fragment carrying the sequence Detect the restriction site

12. Application Diagnosis of human disease Detect point mutation, gene rearrangement or gene amplification Mutated gene  change in the size ( hemophilia A) Gene rearrangement  change in size and pattern ( leukemia ) Amplification  increase in gene copy number (Charcot-Marie-Tooth syndrome)

13. Northern Blotting: 1-Extraction of RNA: - The RNA sample can be: i . total RNA isolated from particular samples ii. RNA containing poly(A) tails, i.e : messenger RNA(mRNA) 2 . Gel Electrophoresis : agarose gel 3. The RNA molecules in the gel are transferred to nitrocellulose or nylon . procedure same for Northern Blotting…………. Alwine adapted Southern's method for DNA to detect, size and quantify RNA – 1977. -No need to digest RNA with restriction enzymes.

14. Northern Blotting

15. Main functions Detect mRNA transcriptional activity Quantifying the transcription Determine the size of the mRNA Determine mRNA level

16. Western Blotting: is an immunoblotting technique which rely on the specificity of binding between the molecule of interest (protein) & a probe ( antibody raised against that particular protein ) to allow detection of molecule of interest in a mixture of many other similar molecules.

17. 1- Sample Preparation: Numerous methods are available for disrupting cells and preparing their contents for analysis by Western blotting

18. 2- Gel Electrophoresis: I-Samples are loaded into separate wells II-Run at 200 volts for 3 0-40 minutes III-Running Buffer ( pH 8.3)

19. 3.Transfere Protein moved from within the gel onto a membrane made of  nitrocellulose by   electroblotting technique . uses an electric current to pull the negatively charged proteins from the gel towards the positively charged anode, and into the nitrocellulose membrane. The entire stack is placed in a buffer solution which moves up the paper by  capillary action , bringing the proteins with it.

20. 4. Labeling with Primary Antibody forms an antibody-protein complex with the protein of interest with specific Antibody. 5. Labeling with Secondary Antibody Is conjugated to HRP (horseradish peroxidase) Acts as antibody against primary antibody Antigens can be visualized through colored reaction 6. Visualization The position of protein of interest is marked by visible band, forming protein-primary antibody-secondary antibody - enzyme complex A flash light is observed which expose x-ray film. The light is due to the release of protons by catalyzing the oxidation luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) by HRP .

21. Western Blotting Gel electrophoresis 1 Electroblotting 2 Labeling with primary antibody 3 Labeling with 2 nd antibody 4 Blocking step Visualization 5

22. Main functions Study a specific gene expression Analyze endogenous protein level Determine the mass of protein Compare with protein molecular weight standards Clinical diagnosis Detect immunogenic responses by infectious agents (bacteria, parasites) E.g. Human immunoglobulin in serum binds to the parasitic proteins that are given externally, indicate parasitic infection