western blot

Westernblot


1. Definition and purpose

Western blot is a method of finding only a desired protein in a protein mixture using an antibody that reacts with the antigen epitope of the protein to be found. Proteins are separated according to size on gel using electrophoresis, and then transferred to membranes such as PVDF and nylon. Detect a specific protein to be found using an antigen-antibody reaction that attaches an antibody (antibody) to which a probe that emits light to membrane is attached. Methods such as fluorescence (fluorescein isothiocyanate), radioactivity, enzyme reaction (peroxidase, alkaline phosphatase, glucose oxidase) are used as a method of looking at only a desired protein among various proteins.


2. Method and principle

1) Preparation of sample

After extracting proteins from cells or tissues using buffers, samples are prepared through protein quantification. Mix the protein sample to be loaded with SDS sample buffer and heat it at 95°C for 5 minutes to release the protein, lose its own charge, and have a (-) charge by SDS. The protein denaturated by the action of SDS passes through the mesh structure formed by acrylamide in a rod-like state. Therefore, the larger the protein, the longer the length, and the longer it takes to get out of the net structure, so the movement speed slows down, and through this, the protein is separated.

At this time, protease or phosphatase inhibitor is added to the buffer that processes the sample to prevent the protein from being destroyed by enzymes in itself.


2) Electrophoresis

Proteins in the sample are separated by electrophoresis according to isoelectric point (isoelectric point), molecular weight (molecular weight), and electric charge (charge). SDS-PAGE is a separation method according to the difference in protein mass, and a buffer solution containing polyacrylamide gel and sodium dodecyl sulfate (SDS) is used. After the sample is denatured by the reducing agent, the protein forms a primary structure and moves, so it can be separated only by the pure molecular weight of the protein.

Since proteins in the sample are coated with SDS with a (-) charge, they pass through a network made of acrylamide and move to the (+) electrode. When a sample and size marker are placed in a well and a voltage is applied to the gel, the protein moves at different speeds, and the small protein moves at a faster speed than the large protein, and the protein is separated according to its size. At this time, the separation ability of the gel is determined according to the acrylamide concentration. The higher the acrylamide concentration, the smaller the hole in the net, so that small molecules can be better separated, and the lower the acrylamide concentration, the larger the hole in the net, so that proteins with a large molecular weight are well separated.

SDA-PAGE


# Stacking gel and Running gel

Usually, acrylamide gels of SDS-PAGE are stacking gels and running gels, and electrophoresis is performed with two different gels attached. The two gels also show a difference in acrylamide content, but the difference in pH is a factor that causes the two gels to play different roles. When an electric field is applied after loading the sample, Cl- and glycine ions contained in the electrophoresis buffer play a major role in the movement of proteins.

Stacking gel

The gel that constitutes the upper part is a large pore polyacrylamide gel (4%). Tris buffer is at pH 6.8, which is lower than the electrophoresis buffer. In the stacking gel, proteins are negatively charged by SDS, Cl- also has a negative charge and has the smallest molecular weight. However, in the case of glycine, since the value where the net charge becomes 0 is pH 6.2, only some of them have a negative charge, so the migration speed is Cl->protein>Glycine. Due to this difference in migration speed between ions, a high voltage gradient is formed, and the migration speed of proteins between the two ions becomes very fast, and since the concentration of acrylamide is also low, the migration speed of the proteins increases and they go down at almost the same speed. As a result, the proteins are located on the same starting line before entering the running gel.

Running gel

This is a polyacrylamide gel with small pores that constitutes the lower part of the gel. Since the Tris buffer uses pH 8.8, glycine is completely ionized this time. Since the migration speed changes from Cl->glycine>protein, the high voltage gradient formed between Cl- and glycine almost disappears, so the migration of proteins is affected by molecular weight, and the acrylamide concentration also increases, so the mesh structure inside the gel becomes denser, so the migration speed of proteins varies depending on molecular weight. Since the separation ability of proteins differs depending on the % of the running gel, it is manufactured by adjusting the % of the running gel according to the size of the protein to be separated. In the current experiment, 8% gel is mainly used to separate 24-205kDa proteins, 10% gel is used to separate 14-205kDa proteins, and 12% gel is used to separate 14-66kDa proteins.

3) Transfer

Proteins isolated on Gel cannot bind antibodies. In order to detect proteins using antibodies, a trnasfer process is required to transfer the protein of gel to a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). At this time, the membrane used is a different membrane depending on the characteristics of the protein to be detected. Attachment of proteins is achieved by electrical interaction between membranes and proteins and hydrophobic bonding.

As shown in the figure, when the kit is mounted in the order of (-) charge - black part of the plastic kit – sponge – filter paper – gel – membrane – paper - sponge – white part of the plastic kit – (+) charge and voltage is applied, protein moves from gel to membrane. This is because the protein uses the property of being (-) charged due to SDS, and the protein is applied to an electric field with the gel side (-) and the membrane side (+) to move from the gel to the membrane. Usually, it runs at 80V for 1 hour, and after transfer, only the membrane is separated and blocking is performed. Methanol is included in the Trasnfer buffer, which helps proteins bound to SDS to bind well to membranes.


After transfer, to check whether the protein has been transferred well to the membrane, stain with a staining reagent such as Ponceau S (within 10 seconds and 1 minute, only slightly enough to show the band) and wash enough with DW or PBS to check the band.

Type	Pros	Cons
Nitrocellulose	- It serves as a moderate support for protein staining or immunochemical characterization. - The price is economical.	- The binding force of the protein is relatively weak. - It is difficult to store for a long time due to its weak material.
Nylon	- It is physically stronger than nitrocellulose and has an excellent ability to adhere to proteins.	- It is difficult to detect the desired protein due to the high back ground.
polyvinylidenedifluoride (PVDF)	- It is relatively excellent in chemical resistance and mechanical force. - It has strong binding power to proteins. - A clean band is formed by a specific combination.	- The price is higher than that of nitrocellulose. - To transfer by hydrorophobic, it should be soaked in methanol in advance and changed to hydrophillic.

Membrane Type

 

 

4) Blocking

When the protein is transferred to the membrane, the protein does not completely cover the memebrane and creates empty spaces in places. Membranes have the property of sticking proteins well, and if the empty space of the membrane is not filled, the non-specific binding of the empty space with the antibody increases the possibility that only the specific protein we want to see will not be detected cleanly. Therefore, a blocking process is needed to reduce the nonspecific binding of antibodies and proteins.

Blocking buffers are generally used by dissolving 3-5% bovine serum albumin (BSA) or non-fat dry milk (skim milk) in a solution containing Tween 20 or Triton X-100 in Tris-bufferd saline (TBS). Blocking buffer is poured to block the membrane, and this blocking buffer is attached to the entire membrane in addition to the place where the protein we want to see is attached, so that only the specific protein we want to see is antibody attached to obtain accurate and clean results.


5) Antibody reaction and detection

Primary antibodies that specifically bind to the protein to be detected are diluted in blocking buffer. The primary antibody is poured to the extent that the membrane is slightly submerged and reacted overnight at 4°C. After the reaction, the membrane is rinsed with a wash buffer (TBS or PBS containing a surfactant) to remove the unbound primary antibody, and then the secondary antibody is reacted with the membrane. The secondary antibody is bound with horse radish peroxidase (HRP), which provides a fluorescent material and allows visual confirmation, and these secondary antibodies bind to the primary antibody to color the protein we want with fluorescence so that it can be confirmed with the naked eye. After the secondary antibody reaction, the secondary antibody that is not bound with the wash buffer is washed again, and the band is checked using a detection reagent such as luminol. HRP oxidizes substrates such as luminol, and bands can be detected through the energy wavelength released at this time.




References
- https://en.wikipedia.org/wiki/Western_blot
- 재미있는 분자생물학 그림여행/ 유욱준, 신인철/ 분자방/ 2009
- http://www.genehunters.co.kr/Training/04.htm
- Protein methods/ Daniel M. Bollag 외/ Wiley-Liss/ 1999