Blotting

The transfer of nucleic acid fragments or proteins from gel to a suitable solid support is known as “Blotting”

Or Biochemical technique in which macromolecules separated on a gel are transferred to a nylon membrane or sheet of paper, thereby immobilizing them for furthur analysis.

Electro-Blotting: an important method to transfer mainly proteins from polyacrylamide gel on to nitrocellulose or other carier membrane. A technique for the electrophoretic transfer of DNA, RNA or protein to a suitable membrane. The method most commonly used for the electrotransfer of proteins to nitrocellulose is that reported by Towbin et al. (1979). This technique was patented in 1989 by William J. Littlehales under the title "Electroblotting technique for transferring specimens from a polyacrylamide electrophoresis or like gel onto a membrane." Transfer of the proteins can be carried out using several methods such as vacuum, capillary or electric field. Electroblotting is by far the most wide-spread technique which utilizes either vertical buffer tanks or semi-dry blotting

Tank-Blotting: The gel & blotting membrane are clamped fram between filter paper & fiber pads. Typically transfer time is overnight, only little transfer buffer is required & transfer time is very short.

Capillary-Blotting: Traditional method for transfer of nucleic acids

• Membrane size up to 20 x 25 cm
• Overnight transfer
• Easy to use

Method for transfer of nucleic acids (especially RNA) from agarose gels onto a membrane. This procedure usually takes up to 12 hours (over night) to complete.

Easy to set up assembly that works without power or vacuum source.

This method is based on the movement of buffer from a reservoir through the gel and the blotting membrane to a stack of dry blotting paper by capillary force. The molecules are carried to the blotting membrane on which they are adsorbed.

Vaccum-Blotting:

Dot & Slot Blotting:

Some of the commercially available blotting products are:

Fastblot B33/34; B43/44; B64

Tankblot Tankbot Ecomini

Vacuum-Blot

Cappilary Blotting

Dot Blot 96/ System

Hybrit Slot

The convertible.

Blot is made in spot/ stain on membrane.  Blotting technique are of various types depending on the targets in the hybridization experiments for their specific detection. Commonly are:

Dot Blotting [For DNA/RNA]

Northern Blotting [For RNA]

Southern Blotting [For DNA]

Western Blotting [For Protein]

Dot blotting

A modification of Southern & Northern blotting techniques where nucleic acids are directly spotted on to the filters and not subjected to electrophoresis. The hybridization procedure is the same as in original blotting techniques.

Sample DNA’s from several tissue/ individual can be tested in a single test run. Dot blot are useful in detecting the presence of the sequence being transferred in a number of suspected transgenic individuals or in different tissues of a single individuals

Steps involve:

Sample DNA/RNA from different individual/tissues are transferred on to a nitrocellulose filter in form of dots;

Denature the DNA & backed filter at 80ºc to fix the DNA firmly on to the filter, that prevent non-specific binding of the probe to the filter;

The filter is then treated with the appropriate radioactive ssDNA probe under condition favoring hybridization;

Wash repeatedly filter to remove the free probe;

Dots having appropriate DNA/RNA sequence will hybridize with the radioactive probes;

The hybridization probes are detected by autoradiography; this denotes the individual/ tissue in which the DNA/RNA sequence corresponding to the probe is represented.

Northern Blotting

The technique for the specific identification of RNA molecules is known as Northern Blotting. This was developed in 1977 by “Alwine et al” at Stanford University.

RNA is separated by RNA gel electrophoresis subsequent transfer to membrane, hybridization with probe & finally detection through autoradiography. RNA molecules don’t easily bind to Nitrocellulose paper/ Nylon membrane. Blot transfer of RNA molecule is carried out by using a chemically reactive paper prepared by diazotization of aminobezyloxymethyl to create diazotibenzyloxymethyl (DBM) paper. The RNA can be conventionally bound to DBM paper.

Steps involve:

Sample target ;

RNA isolation;

Gel electrophoresis of RNA;

Transfer to membrane;

Probe preparation;

Cross-linking of RNA to membrane;

Pre-hybridization;

Hybridization;

Post-hybridization;

Signal detection.

Advantages:

Widely accepted & well regarded method;

A straight forward method;

Often used as a confirmation or check;

Versatile protocol as it can allow the usage of many types of probes, including radiolabeled, non-radiolabeled; invitro-transcribed RNA & even oligo-nucleotides such as Primers.

Disadvantages:

Often radioactivity is used, which prevents ease of performing its use and disposal;

The whole process takes long time;

If RNA sample are even slightly degraded by RNases, the quality of the data and quantization of expression is quite negatively affected.

Application:

Detection of mRNA transcripts size.

Study RNA degradation

Study RNA splicing, can detect alternatively spliced transcripts;

Study internal ribosomal entry site;

Often used to confirm & check transgenic.

Southern Blotting

The technique for DNA separation according size by gel electrophoresis is known as “Southern Blotting” where gel denatures in to single stranded molecules by treatment with alkali, neutralization & transferred to a hybridization method by using a high salt concentration buffer. DNA is then irreversibly bound to the membrane either by heat treatment/ UV cross linking. Thus ssDNA targeted molecules are available on the filter for hybridization with a labeled ssDNA probe. 

This technique, devised by Ed Southern in 1975, is a commonly used method for the identification of DNA fragments that are complementary to a know DNA sequence. Southern hybridisation, also called Southern blotting, allows a comparison between the genome of a particular organism and that of an available gene or gene fragment (the probe).  It can tell us whether an organism contains a particular gene, and provide information about the organisation and restriction map of that gene.

In Southern blotting, chromosomal DNA is isolated from the organism of interest, and digested to completion with a restriction endonuclease enzyme. The restriction fragments are then subjected to electrophoresis on an agarose gel, which separates the fragments on the basis of size.

DNA fragments in the gel are denatured (i.e. separated into single strands) using an alkaline solution. The next step is to transfer fragments from the gel onto nitrocellulose filter or nylon membrane. This can be performed by electrotransfer (electrophoresing the DNA out of the gel and onto a nitrocellulose filter), but is more typically performed by simple capillary action.

In this system, the denatured gel is placed onto sheet(s) of moist filter paper and immersed in a buffer reservoir. A nitrocellulose membrane is laid over the gel, and a number of dry filter papers are placed on top of the membrane. Bycapillary action, buffer moves up through the gel, drawn by the dry filter paper. It carries the single-stranded DNA with it, and when the DNA reaches the nitrocellulose it binds to it and is immobilised in the same position relative to where it had migrated in the gel.

The DNA is bound irreversibly to the filter/membrane by baking at high temperature (nitrocellulose) or cross-linking through exposure to UV light (nylon).

Southern blotting has became a routine technique in the analysis of gene organization, the identification & cloning of specific sequences.

Steps involve:

DNA digestion & electrophoresis;

DNA blotting

Hybridization

Post-hybridization

Autoradiography

Advantages

An invaluable method in gene analysis

For confirmation of DNA cloning results;

Forensic applied to detect minute quantification for DNA.

Western Blotting

The technique identifies proteins involving transfer of electrophorosed protein bands from polyacrylamide gel to nylon or nitrocellulose membrane, detected by specific protein-ligand interaction [i.e. Antibody/ Lectin] 

Western blot identify the location of a specific protein after it has been separated by SDS-PAGE. First a primary antibody that recognizes one epitop binds to the protein of interest. The location of the primary antibody is visualized by adding a secondary antibody conjugated to a detection system.

 Steps involve:

Ø  Separation of protein by Gel-electrophoresis

Ø  Transfer protein to Nitrocellulase membrane so, the protein retain the same pattern of separation they had on the get

Ø  Incubate the blot with a generic protein to binds to any remaining sticky place on the nitrocellulose;

Ø  Photograph, the location of the antibody, colorless substrate attached enzyme converted to colorless product.

In this technique it doesn’t matter whether the protein has been synthesized invivo or invitro, This technique explains the content of protein accumulated in cells and rate of synthesis explained by Radioimmune precipitation assay there is establishment of negative charge on side gel while positive on nitrocellulose membrane. The protein binds better to nitrocellulose membrane at low pH and no air bubbles between nitrocellulose membrane and gel. The primary antibody specific to protein form antibody-protein complex with protein of interest. The protein gel stained with KCl so, form precipitation with an SDS-PAGE gel.

The primary antibody obtained from rabbit antisera dilution with non-fat dry instant milk. The secondary antibody is goat anti-rabbit, antibody against 1^st antibody. The reaction usually run out in about 1 hours.