WESTERN BLOTTING:

BY: SAI MANOGNA (MSIWMO14)

Western blotting, also known as immunoblotting. This method is used for detecting proteins and post-translational protein changes, using antibody-based samples to extract precise protein information from complex samples.

It’s a standard approach in molecular biology, biochemistry, and cell biology with several applications. It can provide semi-quantitative or quantitative protein data in simple or complex biological samples.

Western Blot is also used to separate and identify proteins. In this process, a protein mixture is separated by gel electrophoresis based on molecular weight. These observations are then transferred to a membrane that produces a band for each protein. The membrane is then incubated with protein-specific antibody labels of interest.

The unbound antibody is washed away, leaving the protein of interest with only the bound antibody. By developing the film, the bound antibodies are then detected. Since the antibodies only bind to the protein of interest, there should be only one band visible. The band’s thickness corresponds to the amount of protein present in the sample.

Sample Preparation :

1. Cell lysates are the most common type of sample used in the western blot technique. In the cell cytosol, protein extraction aims to gather all the proteins. This can be achieved with protease inhibitors at a cold temperature to avoid protein denaturing.

2. Using a spectrophotometer, protein concentration is also measured. Using this concentration, the relationship between concentration, density, and volume allows the density of the protein-loaded into each well to be measured.

3. After deciding the necessary sample amount, it is diluted into a glycerol-containing loading buffer so that the samples quickly fall into the gel wells.

4. There is also a monitoring dye (bromophenol blue) in the buffer, allowing the observer to see how far the separation has advanced.

5. After being diluted into a loading buffer, the sample is heated to denature the higher-order structure while maintaining sulfide bridges. High structure denaturing ensures that the negative charge of amino acids is not neutralized, allowing the protein to shift in an electric field.

6. Having positive and negative controls for the sample is also very relevant.

7. A known target protein source, such as a distilled protein or a control lysate, is used for positive control. This helps to validate the protein’s identity and the antibody’s activity.

Gel Electrophoresis :

There are two different kinds of agarose gels used by western Blot: stacking and separating gel.

Stacking gel: The stacking gel is slightly acidic (pH 6.8), which is present at the top position and has a lower concentration of acrylamide, making a porous gel that poorly separates protein, but allows thin, sharply formed bands on the gel.

Separating gel: The separating gel is basic (pH 8.8), also known as resolving gel, which has a higher polyacrylamide content, making the pores of the gel narrower. Therefore, in this gel, the protein is more differentiated by its size, since the smaller proteins migrate more quickly and thus faster than the larger proteins.

The proteins have a negative charge when mounted on the gel since they have been denatured by heating, and when a voltage is applied, they will migrate towards the positive electrode. Using the buffer solution, gels are typically created by pouring them between two glass or plastic plates. Samples are loaded with a marker, and a sample buffer is loaded into the empty wells. The gel is then attached and allowed to run on the power supply. If the voltage is high, it can overheat and distort the bands.

Blotting: After the separation of protein samples, It is then transferred to a membrane.

Nitrocellulose membrane: Two membrane forms exist: nitrocellulose and PVDF. Nitrocellulose is used because of its high protein affinity and retention capacities. It is brittle, however, and does not permit the membrane to be used for reprobation. PVDF membranes provide better mechanical support in this regard and allow reprobation and storage of the Blot. In the PVDF membranes, however, the background is higher; washing is therefore essential.

1. Using an electric field directed perpendicular to the gel surface, the transition is performed, which allows the proteins bands formed to travel out of the gel and onto the membrane.

2. The membrane is embedded into a sandwich between the gel surface and the positive electrode.

3. To cover the gel and blotting membrane, a fiber pad at both ends and a paper towel are used as a sandwich.

Two aspects are essential here:

a. the close contact between the gel and the membrane to ensure a clear image

b. the location between the gel and the positive electrode of the membrane.

For a successful transfer, the membrane has to be placed so that the negatively charged proteins move from the gel to the membrane. This transfer form is called an electrophoretic transfer, which can be achieved in semi-dry or wet conditions. Wet conditions are typically more stable, as the gel is less likely to dry out, and larger proteins are preferred.

Washing, blocking, and antibody incubation :

Blocking is a very significant step in western blotting, as it prevents the non-specific binding of antibodies to the membrane. To minimize the background, blocking is often made with 5 percent BSA or non-fat dried milk diluted in TBST.

Sometimes, non-fat dried milk is preferred as it is readily available and inexpensive. Even milk proteins are not compatible with all detection markers, so the required blocking solution must be selected with caution.

Example- , BSA blocking solutions are preferred to be milk containing casein, a phosphoprotein, and biotin. Incubating the primary antibody with BSA is also a successful idea since it is generally required in higher quantities than the secondary antibody. If the Blot does not provide satisfactory results, placing it in a BSA solution allows the antibody to be reused.

The antibody concentration depends on the sample used. In a wash buffer, such as PBS (Phosphate buffer saline) or TBST (Tris Buffer Saline with Tween 20), the antibody can be diluted. As it minimizes background and removes unbound antibodies, washing is essential. For a very long time, the membrane should not be left to wash, as the signal can also be diminished.

Using the labeled antibody, the membrane is then detected, usually with an enzyme such as horseradish peroxidase (HRP), which is then detected by the signal as it creates corresponding to the target protein’s location. This signal is recorded in a film that is usually made in a dark space.

Quantification :

It is essential to know that data generated with a western blot is usually called semi-quantitative. This is because it offers a relative comparison, though not an absolute quantity calculation, of protein levels.

There are two causes for this; first, there are differences between the samples in separate lanes in the loading and transfer rates that are different on independent blots. Until a more reliable comparison can be made, these variations would need to be standardized. Second, the detection-generated signal is not linear across the sample concentration spectrum. Therefore, because the produced signal is not linear, the concentration model should not be used.

Fig: Sequential stages of the Western blot process.

REFRENCES:

Photo courtesy of; http://onlinelibrary.wiley.com/doi/10.1111/sms.12702/full

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