Southern, Northern and Western blotting technique
Blotting:-
> It is the separation and transfer of macromolecules onto a blotting membrane to detect and identify such macromolecules, as DNA, RNA or Protein.
> 4 types:-
i. Southern blotting:- Detect DNA.
ii. Northern blotting:- Detect RNA.
iii. Western blotting:- Detect Protein.
iv. Eastern blotting:- Detect Protein.
General Procedure:-
- The target molecule in a sample is isolated.
- Electrophoresis separates the molecules.
- The separated contents are transferred onto a membrane. This process is celled blotting.
- The membrane is then exposed to radiolabeled probes and incubated. Probe binds to the target molecule.
- The probe and target molecule create bands that can be visualized with X-ray film.
1. Southern blotting:-
- Discovered by Edward M. Southern at Edinburgh University in 1970.
- It is designed to locate a particular sequence of DNA within a complex mixture. For example, it could be used to locate a particular gene within an entire genome.
Procedure:-
a. Digestion:- Digest the DNA with an appropriate restriction enzyme.
b. Electrophoresis:- Run the digest on an agarose gel.
c. Denaturation:- Denature the DNA on the gel. For this soak it in about NaOH, which would separate double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.
d. Depurination:- Fragments greater than 15 kb are hard to transfer to the blotting membrane. Depurination with HCl takes the purines out, cutting the DNA into smaller fragments. Neutralize the acid after this step.
e. Blotting:-
- It is the transfer the denatured DNA to the membrane.
- A nitrocellulose membrane or nylon membrane is used. Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm.
- Many scientists feel nylon is better since it binds more and is less fragile.
- Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.
- You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC (Saline Sodium Citrate) through the membrane. This works similarly to capillary action, except more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)
- After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)
f. Hybridization:-
- It is the process of annealing the probe to the DNA on the membrane due to complementarity.
- Probing is often done with:
i. 32P labeled ATP
ii. Biotin / Streptavidin
iii. Bioluminescent probe
g. Visualization:-
- Visualize your radioactively labeled target sequence.
- If you used a radiolabeled 32P probe, then you would visualize by autoradiograph.
- Biotin / Streptavidin detection is done by colorimetric methods.
- Bioluminescent visualization uses luminesence.
2. Northern Blotting:-
- The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.
- Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern.
- It is designed to locate a particular sequence of RNA within a complex mixture.
Procedure:-
a. Extraction of RNA:- There are many RNA extraction kits commercially available, but they all involve cell lysis, inhibition of RNAases, removal of proteins and other contaminants, and recovery of RNA.
b. Isolation of mRNA:-
- Oligo dT cellulose chromatography can be used to isolate only mRNA with a polyA tail. The poly A tail is the final step of mRNA production in the nucleus. The tail enables nuclear export, translation, and stability of mRNA.
- In Oligo dT cellulose chromatography, oligos complementary to the poly A tail are covalently attached to a resin column.
- When the sample is applied to the column the mRNA with the poly A tail will hybridize to the oligo probe and be retained on the column.
- Then, the elution buffer is applied to disrupt hybridization and recover the mRNA.
c. Gel electrophoresis to separate mRNA by size:-
- Agarose gels containing formaldehyde were traditionally used to denature RNA.
- The formaldehyde reacts with the imine and amine groups on the nucleic acids, which disrupts the hydrogen bonding between bases and disrupts the secondary structure of the RNA.
- It is important to disrupt the secondary structure because the RNA must be extended to allow proper binding of probe for identification.
d. Transfer of RNA to blotting membrane:-
- The transfer is necessary because the probes can’t enter into the gel matrix. Therefore, the RNA must be transferred to a membrane where they can be accessed by the probes.
- Transfer is accomplished via a capillary (overnight) or vacuum (15-60 minutes) blotting system.
- The blotting membrane is positively charged to attract the negatively charged RNA. Nylon is a commonly used membrane.
v. Immobilization of RNA to the blotting membrane:- Covalently attached to the membrane by the application of UV light or heat.
vi. Application of Probe:-
- Probes have a minimum of 25 bases that are complimentary to the mRNA sequence of interest.
- Excess probe is washed off.
vii. Probe visualization:-
- Radioactive isotopes were traditionally used, but have been replaced in favor of safer detection methods.
- Chemiluminescence is commonly used in the modern northern blot protocol.
3. Western Blotting:-
Principle:-
> Western blot is performed by using polypropylene gel electrophoresis. SDS-PAGE allows protein samples to be separated and transferred to a solid support, such as nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane.
> The solid support can absorb the protein and keep its biological activity unchanged. The transferred solid support membrane is called a blot and is treated with a protein solution to block the hydrophobic binding site on the membrane.
> The membrane is treated with the antibody (primary antibody) of the target proteins.
> Only the proteins to be studied can specifically bind to the primary antibody to form an antigen-antibody complex.
> After the primary antibody is washed and removed, only the position of the target protein binds to the primary antibody.
> The primary antibody-treated membranes are treated with a labeled secondary antibody after washing.
> After treatment, the labeled secondary antibody that binds to the primary antibody forms an antibody complex that can indicate the location of the primary antibody, both the location of the protein being studied.
Procedure:- There are six steps involved in western blot -
i. Sample preparation:-
> Proteins can be extracted from different samples, such as tissues or cells.
> Since tissue samples display a higher degree of structure, the tissues are first broken down by the mechanical invention, such as homogenizer or sonication.
> Protease and phosphatase inhibitors are commonly used to prevent the digestion of the sample at cold temperatures.
> After protein extraction, it is important to detect the concentration of proteins, which permits the mass of proteins loaded into each well. And a spectrophotometer is often used for proteins concentration.
ii. Gel electrophoresis:-
> The most commonly used gel is polyacrylamide gels (PAG) and buffers loaded with sodium dodecyl sulfate (SDS).
> Western blot uses two types of agarose gel:
Stacking gel:- that is used for concentrate all proteins in one band
Separating gel:- that allows for separating proteins according to their molecular weight.
> Smaller proteins migrate faster in SDS-PAGE when a voltage is applied.
> PAGE can separate proteins ranging from 5 to 2,000 kDa according to the uniform pore size which is controlled by the Different concentration of PAG.
> Typically separating gels are made in 5%, 8%, 10%, 12% or 15%.
> When we choose the appropriate percentage of the separating gel, we should consider the size of the target proteins.
> The smaller the known weight of proteins is, the higher percentage of gels should be used.
iii. Proteins transfer:-
> After separating proteins by gel electrophoresis, proteins are moved from within the gel onto a solid support membrane to make the proteins accessible to antibody detection.
> The main method for transferring proteins is called electroblotting, which uses an electric field oriented perpendicular to the surface of the gel, to pull proteins out of the gel and move into the membrane.
> It can be done semi-dry or wet conditions, while wet conditions are usually more reliable as it is less likely dry out the gel.
> The membrane is placed between the gel surface and filter. The transfer sandwich is created as follows: a fiber pad (sponge), filter papers, the gel, a membrane, filter papers, a fiber pad (sponge).
iv. Blocking:-
> Blocking is an important step in the western blot to prevent antibodies from binding to the membrane non-specifically.
> The most commonly used typical blockers are BSA and non-fat dry milk.
> When the membrane is placed in the dilute solution of proteins, the proteins attach to all places in the membrane where the target proteins have not attached. In this way, the “noise” in the final product of the western blot can be reduced and result in clearer results.
v. Antibody incubation:-
> After blocking, the primary antibody binds to target protein when the primary antibody is incubated with the membrane.
> The choice of a primary antibody depends on the antigen to be detected.
> Washing the membrane with the antibody-buffer solution is helpful for minimizing background and removes unbound antibodies.
> After rinsing the membrane, the membrane is exposed to the specific enzyme conjugated secondary antibody.
> When performing secondary antibody incubation, the labeled secondary antibody can bind to the primary antibody which has reacted with target proteins.
> Based on the species of the primary antibody, we can choose the appropriate secondary antibody.
vi. Protein detection and visualization:-
> A substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance. It enables us to know the densitometry and location of the targets protein. And the size approximations are taken by comparing the proteins bands to the marker.
> There are several detection systems are available for protein visualization, such as colorimetric detection, chemiluminescent detection, radioactive detection, and fluorescent detection.
> The electrochemiluminescence (ECL) system is the most common detection method.