Western Blotting is a qualitative and semi-quantitative analytical technique used to detect a specific PROTEIN present in a particular sample. It is semi-quantitative because it gives us a rough estimation of target protein concentration and not the exact amount of the detected protein. This particular sample is mostly a mixture of different proteins extracted from cell, tissue, bacteria or virus. By using this technique, bio-technologists are able not only able to know absence or presence of a protein but also have an idea of the concentration of a specific protein, hence semi-quantitative (Because it does not tell us the exact concentration rather give a rough idea of the quantity of the proteins we are looking for). Four main steps are involved in western blot:
- Sample preparation by
lysis – to extract the proteins from cell/Tissues - Gel electrophoresis
(SDS-PAGE) – to separate the proteins on the basis of their weights - Blotting – To
transfer the proteins from within the sample onto a membrane - Detection – probing
the proteins with antibodies tagged with a reporter molecule
Principle of Western Blot
First of all, proteins are extracted from any source mostly by lysing the cell and separating the protein from cell debris by centrifugation. Secondly, this mixture of proteins go through gel electrophoresis to separate the protein on the basis of their size, hence type. Thirdly, these protein bands present on the gel is transferred to a membrane, this step is called blotting. Finally, this membrane is treated with antibodies specific to our target protein. Since, antibodies used in western blotting are labelled with enzymes, fluorophores, radioisotopes or gold-conjugation that lead us to the detection of the target protein.
Sample preparation – Cell lysis, centrifugation, concentration optimization
Proteins that are to be detected are usually extracted from cell or tissue. Proteins are extracted from a cell or tissue by breaking the cell wall (cell lysis). After cell lysis, centrifugation is done to extract the protein from the sample. If proteins are extracted from the cell, the resulting product is called cell lysate; and when proteins are extracted from tissue, it is called tissue homogenate (fig. 01).
The concentration of protein is measured using a spectrophotometer. When the concentration is known, the sample is prepared and concentration is adjusted and optimized for the next step of Western blot i.e. gel electrophoresis.
- To aid cell lysis different
buffers, detergents and salts are used. - To prevent the digestion of
the sample different anti-proteases and phosphatases are used. - To avoid protein
denaturation, tissue or cell lysis is performed at a low temperature.
Gel Electrophoresis to separate the proteins
Sample containing proteins is loaded into the wells in the SDS-PAGE gel. The sample is taken in micrograms (20-30ug) if we you are dealing with the cell lysate or tissue homogenate and in nanogram in case of pure proteins. Equal amounts of proteins are loaded into each well .One well is reserved for the ladder. The ladder is a mixture of pre-stained pure proteins with their known molecular weight. When the samples are loaded in each well, voltage is applied to the gel. Proteins being negatively charged because of the SDS, migrate towards the positive terminal of the electric supply. (fig 02).
Smaller proteins move
faster while larger proteins move slower than relative smaller proteins in a
specific period of time. The reason behind this difference in the
electrophoretic mobility of proteins is the size of each protein. Acrylamide
concentration determines the resolution of SDS-PAGE gel. The greater the
acrylamide concentration, the better the resolution of lower molecular weight
proteins. The lower the acrylamide concentration, the better the resolution of
higher molecular weight proteins. This difference in the electrophoretic
mobility separates the proteins in the gel leading to the formation of
invisible bands in the gel. Bands of the proteins are still not visible at this
stage because no colored or fluorophore is present. The only ladder is visible because it was pre-stained.
Blotting
The transfer of the protein band from within the gel onto a membrane is referred to as blotting. The membrane is placed in direct and close contact with the SDS-PAGE gel. Proteins move from the gel onto membrane maintaining their original position and concentration. The transfer of protein onto membrane is done either by capillary action (older, slower) or by an electric current – electroblotting (latest, faster). The membrane used for blotting is made of either nitrocellulose or PVDF (polyvinylidene difluoride). Each membrane used in Western Blotting has its own benefits; the nitrocellulose membrane is cheap but delicate and cannot stand repeated blotting. On the other hand, PVDF is costly but can be reused after washing off the proteins.
The membrane is placed onto the gel, this gel-membrane layer is sandwiched between the layers of filter papers to protect the gel and membrane. A sponge is placed on each side of this sandwich. The whole sandwich now called “transfer sandwich” is dipped in the buffer solution (known as “transfer buffer”) and electric current is applied ensuring that membrane is present between the gel and positive terminal of the electric supply (fig 03). On applying the electric current, negatively charged proteins move towards the positive terminal of the electric supply. On their way to the positive terminal, they bind to the membrane while maintaining the organization they had within the PVDF/Nitrocellulose gel. The forces responsible for the non-specific binding is hydrophobic interaction and charge interaction present between the membrane and proteins. The time required for the transfer is directly proportional to the thickness of the gel. E.g. it takes 45min for 0.75mm gel. At this stage, all the proteins are now bound to the membrane but not visible. Still, the only ladder is visible on the membrane.
Blocking
As the membrane is specially chosen for its binding to all proteins, both target proteins and antibodies (immunoglobulin proteins) will bind to the membrane on all places. To avoid the non-specific binding of the antibodies onto the membrane, the membrane is dipped in a blocking solution. Blocking solution contain proteins that bind to all places except where target proteins are already bound (fig 04).
Blocking solution can be:
- 3–5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) + 0.1% of detergent such as Tween 20 or
Triton X-100 - 3–5% non-fat dry milk in Tris-buffered saline (TBS) + 0.1% of detergent such as Tween 20 or Triton X-100
Then this membrane is washed with buffer to remove the extra blocking solution. At this stage, imagine
the membrane full of proteins bound to it. Some proteins are target proteins
that are present in their respective
position. Most proteins are from blocking solution. Thus, when antibody
solution is poured onto the membrane, antibody attaches to its specific target
protein only. This blocking step reduces the background in the final result and
also eliminates the chances of false positives.
Detection
Target
proteins are detected by antibodies labelled with a reporter molecule. A
reporter molecule can be
- An enzyme that changes its chromogenic substrate (soluble color)
into an insoluble color product. (Colorimetric Detection) - An enzyme that changes its substrate (luminol) into product +
light. (Chemiluminescent Detection) - A fluorescent dye that fluoresces at when exposed to
a particular wavelength and can be detected by imaging the blot.( Fluorescent Detection) - Radioactive isotopes – radiations from a radioactive molecule such as radioactive iodine
molecule are detected by placing the photographic film onto the membrane. Black
bands appear on photographic film that correspond
to protein bands. (Radioactive Detection)
Two Step detection (Indirect
Detection)
Two types of antibodies are used. The
first antibody (Primary Antibody) is specific to target protein and the second
antibody (Secondary antibody) tagged with reporter molecule binds to the first
antibody.
The membrane is dipped with primary
antibody solution and incubated for 30 min. The primary antibody solution is
discarded and the membrane is quickly rinsed with wash buffer. Now secondary
antibody solution is poured onto the membrane and incubated for 30min while
shaking. Protein bands appear (in case of Colorimetric Detection) on adding the substrate for the
enzyme which is tagged on secondary antibody (Fig
05).
One Step Detection (Direct Detection)
Only one antibody is used which is not
only specific to target protein but also tagged with a reporter molecule.
The membrane is dipped only in one solution of primary antibody. The bands of the proteins on the membrane appear when substrate is added for the enzyme that is tagged onto primary antibody. (Fig 05).
