BLOTTING TECHNIQUES

Dr.Ramesh C.K. Associate Professor and Chairman Department of Biotechnology Sahyadri Science College Shimoga - 577 203 Karnataka, India BLOTTING TECHNIQUES

Southern Blot Northern blot Western blot

It is used to detect It is used to detect It is used to detect the DNA. the RNA. proteins. SOUTHERN BLOTTING A technique for identifying specific sequences of DNA in which DNA fragments are separated by electrophoresis, transferred to a membrane, and identified with a suitable probe. Detects restriction fragments following a restriction enzyme digest of genomic DNA (RFLPs)

Named after British biochemist Edwin Southern (alive and publishing!) Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol Biol. 1975 Nov 5;98(3):503-17. History/Background

• ‘Southern’ hybridization named after Sir Edwin Southern • Developed in 1975 • One of the most highly cited scientific publications “Used to detect the DNA” • This method Involves:  Separation  Transfer  Hybridization. • This DNA can be: • Single gene • Part of a larger piece of DNA……..viral genome “The key to this method is Hybridization” Hybridization

“Process of forming a dsDNA molecule between a ssDNA probe and a ss-target DNA”

PRINCIPLE

 The mixture of molecules is separated.  Immobilized on a matrix.  Probe addition to the matrix to bind to the molecules.  Unbound probes are removed.

“The place where the probe is connected corresponds to the location of the immobilized target molecule.” Steps

The DNA is digested

Fragments

Gel electrophoresis

Transfer to membrane

Probing

Autoradiogram I. DNA Purification – Isolate the DNA in question from the rest of the cellular material in the nucleus. – Incubate specimen with detergent to promote cell lysis. – Lysis frees cellular proteins and DNA. SOUTHERN BLOTTING

• Proteins are enzymatically degraded by incubation with proteinase. • Organic or non-inorganic extraction removes proteins. • DNA is purified from solution by alcohol precipitation. • Visible DNA fibers are removed and suspended in buffer. SOUTHERN BLOTTING

II. DNA Fragmentation – Cut the DNA into different sized pieces. – Use restriction endonucleases (RE) – Bacterial proteins – In vivo, they are involved in DNA metabolism and repair or in bacterial host defense. Step 1. Restriction Enzyme Digestion SOUTHERN BLOTTING

• This allows for specific sequences to be identified more readily. • Fragments are now easily separated by gel electrophoresis. SOUTHERN BLOTTING

III. Gel Electrophoresis – Sorts the DNA pieces by size – Gels are solid with microscopic pores – Agarose or polyacrimide – Gel is soaked in a buffer which controls the size of the pores – Standards should also be run SOUTHERN BLOTTING

• Nucleic acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size. Gel Electrophoresis

_ + Gel Electrophoresis

_ + SOUTHERN BLOTTING

• Gels can be stained with ethidium bromide. • This causes DNA to fluoresce under UV light which permits photography of the gel. • You can tell the exact migration of DNA standards and the quality of the RE digestion of the test DNA. Step 4. Transfer DNA to Membrane Goals of Southern Hybridization

• Blotting Transfer the DNA from the gel to a solid support. Immobilize DNA onto a permanent substrate . ‘Membrane’ – paper-like matrix nitrocellulose paper – Or nylon usually has a slight positive charge SOUTHERN BLOTTING

• DNA is partially depurinated with dilute HCL which promotes higher efficiency transfer by breaking down fragments into smaller pieces. • DNA is then denatured with an alkaline solution such as NAOH. • This causes the double stranded to become single-stranded. DNA Denaturation

• Eliminate hydrogen bonds with sodium hydroxide (NaOH)

A C T T G A

T G A A C T SOUTHERN BLOTTING

• DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe. • Transferred by either electroblotting or capillary blotting.

Transfer DNA to Membrane • Two methods for transferring DNA to a membrane – capillary – Electrophoretic/electroblotting SOUTHERN BLOTTING • 1) Electrophoresis- takes advantage of the molecules negative charge. SOUTHERN BLOTTING

• 2) Capillary blotting-fragments are eluted from the gel and deposited onto the membrane by buffer that is drawn through the gel by capillary action. SOUTHERN BLOTTING

• The blot is made permanent by: – Drying at ~80°C – Exposing to UV irradiation Pre-hybridization V. Blocking Buffer binds to areas on the blot not occupied by • target DNA. Blocks the empty sites from being bound during hybridization. Prehybridization buffers contain ‘blocking reagents’ that occupy available binding sites on the membrane Probes Short sequence of nucleotides which can bind to target sequence. Synthetic oligo nucleotides Should be specific, complementary to target sequence Probe Size- 10 -10,000 bases Most common 14 – 40 bases Long Probes –More stable Takes longer time to hybridize Short Probes – Rapid hybridization, SOUTHERN BLOTTING

• VI. Preparing the probe – Small piece of DNA used to find another piece of DNA – Must be labeled to be visualized – Usually prepared by making a radioactive copy of a DNA fragment. SOUTHERN BLOTTING

• The DNA fragment is labeled by the Random Hexamer Labeling Process: – 1. The template DNA is denatured by boiling. – 2. A mixture of hexamers (6 nucleotides) containing all possible sequences is added and allow to base pair. – 3. DNA polymerase is added with radioactive nucleotides. – 4. The mixture is boiled to separate the strands and is ready for hybridization. SOUTHERN BLOTTING

• The Random Hexamer Labeling Process produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe.

Probe

• How do we detect the probe? – Radioactivity (32P) Probe

• How do we detect the probe? – Digoxigenin (DIG)

U SOUTHERN BLOTTING

• VII. Hybridization – The labeled probe is added to the blocked membrane in buffer and incubated for several hours to allow the probe molecules to find their targets. Hybridization Hybridization

SOUTHERN BLOTTING

• VIII. Washing – Excess probe will have bound nonspecifically to the membrane despite the blocking reagents. – Blot is incubated with wash buffers containing NaCl and detergent to wash away excess probe and reduce background. Washes Anti-DIG Anti-DIG Washes Step 11. CSPD Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2´-(5'-chloro)tricyclo [3.3.1.13,7]decan}-4-yl)phenyl phosphate Step 12. Detection

• DIG-labeled probes emitting minute amounts of light • Digoxigenin (DIG), which is used to label the probe, is widely used for immune detection. This plant steroid molecule is highly antigenic. Anti- digoxigenin can be used to very specifically detect the digoxigenin on a hybridised blot. • To be able to visualise the (polyclonal) DIG antibodies when they are bound to the digoxigenin, they have been covalently linked to the enzyme alkaline phosphatase (AP). • (chemiluminescence) • 32P-labeled probes emitting β- particles Step 12. Detection

• DIG-labeled probes emitting minute amounts of light (chemiluminescence)

• 32P-labeled probes emitting ß-particles

• Autoradiography film can detect this radiation

Making A Southern Blot 4 Probe Hybridization Addition of blocking reagent Probe addition After washing

Membrane with Parts of the bound DNA membrane not already covered Probe covers the with DNA now membrane, but only binds to Probe only remains bind blocking annealed to reagent complimentary DNA complimentary DNA Making A Southern Blot 5 Autorad Development

Membrane with probe bound to complimentary DNA X-ray film is placed over the membrane and left until radiation Fragments from the probe has complimentary to the exposed the film probe appear as bands on the autorad USES

• Identify mutations, deletions, and gene rearrangements • Used in prognosis of cancer and in prenatal diagnosis of genetic diseases • Leukemias • Diagnosis of HIV-1 and infectious disease USES

• Every person has repeated sequences of base pairs which are called Variable Number Tandem Repeats (VNTRs) • To find a particular VNTR we use a radioactive version of the one in question. • This pattern is known as a DNA fingerprint. USES

• Applications of DNA fingerprinting include: – Paternity and Maternity Testing – Criminal Identification and Forensics – Personal Identification NORTHERN BLOTTING

James Alwine, David Kemp, and George Stark at Stanford University in 1979. In this technique RNA is separated by gel electrophoresis. . Southern’s technique has been of enormous value, but it was thought that it could not be applied directly to the blot-transfer of RNAs separated by gel electrophoresis, because RNA was found not to bind to nitrocellulose. . Alwine et al. (1979) therefore found a procedure in which RNA bands are blot-transferred from the gel on to chemically reactive paper, where they are bound covalently..

61 Conti… . The reactive paper is prepared by diazotization of aminobenzyloxymethyl paper (creating diazo benzyloxy methyl (DBM) paper).

. It is prepared from Whattman 540 paper by a series of uncomplicated reactions.

. Once covalently bound, the RNA is available for hybridization with radiolabelled DNA probes.

. As before, hybridizing bands are located by autoradiography.

62 Conti… • Subsequently it was found that RNA bands can indeed be blotted on to nitrocellulose membranes under appropriate conditions (Thomas 1980) and suitable nylon membranes have been developed.

• Because of the convenience of these more recent methods, which do not require freshly activated paper, the use of DBM paper has been superseded.

63 Northern blotting

The RNA samples are most commonly separated on agarose gels containing formaldehyde as a denaturing agent for the RNA to limit secondary structure Steps involved in N.B RNA isolation

Loading of sample on Agarose gel

Blotting on nitrocellulose membrane

Labeling with probe

Washing to remove unbound probe

Detection by autoradiogram

APPLICATIONS

• Northern blot analysis reveals information about RNA identity, size, and abundance, allowing a deeper understanding of gene expression levels. • Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment.

• - A standard for the direct study of gene expression at the level of mRNA (mRNA transcripts) • Detection of mRNA transcript size • Study RNA splicing - can detect alternatively spliced transcripts • Study RNA half-life • Northern blot analysis can also be used to detect cancerous pancreatic cells and tissues. • The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue, as well as the gene expression in the rejection of transplanted organs. Advantages and disadvantages

.Analysis of gene expression can be done by several different methods including RT-PCR, RNase protection assays, microarrays, serial analysis of gene expression (SAGE), as well as northern blotting. .Microarrays are quite commonly used and are usually consistent with data obtained from northern blots; however, at times northern blotting is able to detect small changes in gene expression that microarrays cannot. .The advantage that microarrays have over northern blots is that thousands of genes can be visualized at a time, while northern blotting is usually looking at one or a small number of genes. .The advantages of using northern blotting include the detection of RNA size, the observation of alternate splice products, the quality and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and reprobed for years after blotting. WESTERN BLOTTING Western blotting

• Also called immunoblotting because an antibody is used to specifically detect its antigen • The method originated in the laboratory of Harry Towbin The name western blot was given to the technique by W. Neal Burnette and was developed by George Stark

“A technique in which proteins are separated by gel electrophoresis and transferred to a membrane sheet. A specific protein is then identified through its reaction with a labeled antibody.” Principle

This technique works on the principle on “Antigen-Antibody” relationship Prerequisite for W.B

The SDS PAGE technique is a prerequisite for Western blotting.

“SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it.” Steps in W.B

1. Gel electrophoresis: The proteins are separated according to size.

2. Membrane Transfer: Transferring to nitrocellulose or polyvinylidene difluoride (PVDF) by applying current.

3. Blocking: Done to prevent non-specific protein interactions between the membrane and the antibody protein. Western n blotting

: separates the components according to their molecular weight.

: the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current.

: probed with Ab & then radiolabeled or enzyme- linked 2nd Ab.

: a position is visualized by means of an ELISA reaction.

Cont… Blocking in W.B • The blot is incubated with a generic protein (such as milk proteins) which binds to any remaining sticky places on the nitrocellulose. • Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically 3- 5% Bovine serum albumin (BSA) or non-fat dry milk in Tris-Buffered Saline (TBS), with a minute percentage of detergent such as Tween 20 or Triton X-100. • After blocking, An antibody that is specific for the protein of interest(the primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen. • incubated with the membrane under gentle agitation., the solution is composed of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk or BSA. • The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight. • Agitation of the antibody is recommended to enable adequate homogenous covering of the membrane and prevent uneven binding. Cont..

• Following several rinses for removal of non-specifically bound Ab1, the Ab1-antigen complex on the nitrocellulose sheet is incubated with a second antibody (Ab2), which recognizes the primary antibody and binds it. Secondary antibody

.The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhance the signal.

.Most commonly, a horseradish peroxidase-linked secondary is used to cleave a chemiluminescent reagent, and the reaction product produces luminescence in proportion to the amount of protein.

.A sensitive sheet of photographic film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies bound to the blot. Radioactive detection Radioactive labels do not require enzyme substrates, but rather, allow the placement of medical X-ray film directly against the western blot, which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest

Chemiluminescent detection Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by CCD cameras which capture a digital image of the western blot or photographic film.

Fluorescent detection

The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as a CCD camera equipped with appropriate emission filters which captures a digital image of the western blot Applications

Western Blot Applications / Western Blotting Applications • In terms of western blotting applications, the technique can be employed in both scientific research and medical diagnosis. Western Blot Applications for Research Use In the application of scientific research, it is often used to determine whether the target protein has been successfully expressed or is present in a given cell or tissues and how much of the protein may be present. • Western blot can identify the nature of the protein or epitope effectively. • Applications in epitope mapping, amino acid composition and sequence analysis, spots imprinting analysis, structure domain analysis, and so on. Western Blot Applications for Medical Diagnosis • The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. The most widely used application of this procedure is in confirmatory testing for HIV, where Western blotting is used to determine whether the patient has antibodies that react with one or more viral proteins. • A western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease'). • Some forms of Lyme disease testing employ western blotting. • Western blot can also be used as a confirmatory test for Hepatitis B infection. • Western Blot for Herpes