Brief Introduction: Blotting Techniques
Blotting is used in molecular biology for the identification of proteins and nucleic acids and is widely used for diagnostic purposes. This technique immobilizes the molecule of interest on a support, which is a nitrocellulosic membrane or nylon. It uses hybridization techniques for the identification of the specific nucleic acids and genes.
The blotting technique is a tool used in the identification of biomolecules such ad DNA, mRNA and protein during different stages of gene expression. Protein synthesis involves expression of a DNA segment which gets converted to mRNA to produce the respective protein.
Subtypes of blotting such as northern, western & southern depend upon the target molecule that is being sought. When a DNA sequence is the foundation or code for a protein molecule, the particular DNA molecule of interest can be blotted using Southern Blotting technique. During gene expression, when the DNA is expressed as mRNA for a protein production, this process can be identified by Northern blotting. Finally, the coded mRNA produces the concerned protein, this protein identification can be done by Western Blotting.
General Procedure for blotting
- Homogenize the sample.
- Separation of the molecule of interest by an electrophoresis membrane.
- Transferring the molecules to a nitro cellulosic membrane/ nylon membrane.
- Hybridization or identification of the molecule
Western blotting is the technique used for separation or identification of protein molecules. This technique can be used for both the active 3D protein and denatured long peptide chains. The 3-D protein in its active structure has sulfur-hydroxyl bonds in the structure. This methodology classifies protein based on the molecular weight and charge. These techniques have application in the identification of a wide variety of infectious diseases like HIV, Hepatitis B, Herpes type 2, feline immunodeficiency disease. The identification of these diseases is done by using the antibody of the particular disease as probe and these probes are produced in vitro condition. Western blotting is also used for research purpose. This technique can be used in the study of the properties and activity of a protein molecule of interest.
Gel electrophoresis is the first part of the western blotting technique. For this step two types of gel are used i) Poly Acrylamide Gel Electrophoresis (PAGE) and ii) Sodium Dodecyl Sulfide (SDS). PAGE is used for proteins which have uniform negative charge. SDS is used in case of 3D protein which contains positive and negative charged amino acid groups. The function of SDS is used to give a uniform negative charge to the protein. SDS is both used as the buffer and in the gel based on the protein molecule of interest. When SDS is used gel making, the electrophoresis technique is known as SDS- PAGE electrophoresis. At isoelectric point, the protein molecule acquires a certain charge based on pH. At this point when a constant voltage is applied the protein molecules move towards the electrode. This property leads to the separation of the protein molecule. The size of the protein molecule hinders the rate of movement. So molecular weight also acts as a factor for separation rate in electrophoresis. Based on these two factors, electrophoresis is classified as 1D and 2D. In 1D protein molecules move in the singular axis based on both molecular weight and charge while in 2D protein moves in two axes.
Once the electrophoresis is done the next step is blotting transfer. Blotting is carried out by two mechanisms. Electroblotting uses electric energy to pull out the protein molecule from the gel to the blotting membrane. Capillary blotting uses passive transport of protein molecules together with water molecules as water high concentration moves to lower concentration. Capillary blotting takes a lot of time so this technique is rarely used these days. Once blotting is done the next step is blocking. Blocking is the stopping or arresting of nonspecific antibody interaction. When an antibody is used as a probe it can bind with the gel molecules. This is stopped by the dipping the blotting membrane in a dilute solution of protein Many blocking agents are present in the market ex non-fat dry milk, 3-5% bovine serum albumin,etc.,
Incubation is the final step in the western blotting technique. This probing process is carried in two steps. The primary antibody has the binding capacity with the protein of our interest. The primary antibody is probed into the blotting membrane. Next the secondary antibody is attached to the part of the primary antibody. This secondary antibody has identification characteristics. It can have horseradish peroxidase which is used in the colorimetric method or radioactive component attached to it.Detection of the protein of the interest can be done colorimetric, chemiluminescence or radiography methods.
- Add 5 mL of cold Phosphate Buffered Saline (PBS) to the cell culture or sample. Gently disperse the cell solution. Discard PBS.
- Add PBS and centrifuge at 1500 RPM for 5 minutes. This is known as lysed cell solution.
- Pipette out 180 micro L of lysis solution to 20 micro L of protease inhibitor. This prevents the protease enzyme. Incubate for 30 minutes.
- Centrifuge this solution for 30 minutes at 12000 RPM at 40 c and the sample solution is ready.
10% gel stacking solution is used generally. It is cast into the assembly and given 30 minutes to polymerize and form the gel. The gel has two parts stacking gel and separation gel.
Run the gel at 40 volts until the sample reaches the stacking gel and changed into 80 volts from the separation gel. Protein markers are used in one extreme of the gel.
Fixing and blotting
Fixing is done 5% of bovine serum albumin solution. Blotting is carried in two ways, capillary blotting or through electroblotting. Usually, electroblotting is carried out at 40 volts. For capillary blotting, the gel is stacked in the following order: electrophoretic gel followed by blotting membrane followed by wet tissue and lid glass plate.
The primary antibody is introduced and it is incubated for 30 minutes. Then the unbinding ends are again blocked by BSA and washed. The secondary antibody is added to the blotting plate. It is also incubated for thirty minutes followed by fixing by BSA wash.
Detection and identification can be carried out by a number of methods like radiography, chemiluminescence, colorimetric and x ray methods.