Structural Biochemistry/Proteins/Western Blotting

Western Blotting is another technique that utilizes antibodies to detect the presence of a specific protein in a sample. It is capable of detecting small amounts of protein within a cell or body fluid. Western blotting grants the ability to find proteins in a mixture.

Western blotting, which originated from the laboratory of George Stark at Stanford, is the technique for detecting a particular protein by staining with specific antibody. Its analysis can give us the information about the size of the protein or how much protein accumulated in cells. It does not mean that it can do for any protein since in the case the protein is degraded quickly; it is hard to detect it well. The important thing in this technique is antibody since Western blotting is based on the use of a “quality” antibody as a probe to detect specific protein. In this technique, first we separate the protein by using SDS-poly-acryl-amide gel electrophoresis, which base on the protein's size. Later, those proteins on the gel are transferred to the surface of a polymer sheet by blotting. As soon as we add antibody that is specific for the desired protein to the sheet, it will bind to this protein since they have nothing else to bind to. Later, we wash unbound primary antibody, and this protein-primary antibody can be detected by secondary antibody. Those secondary antibody can only recognize the primary antibody and many of them bind to one primary antibody and give out the signal, which is a radioactive label on the secondary antibody produces a dark band on X-ray film.

Techniques



 * 1) Sample undergoes SDS PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) in which SDS detergent is attached to proteins, giving them negative charge relative to size so that proteins migrate to positive charged cathode. Larger proteins move slower through the acrylamide gel matrix whereas smaller proteins can move faster resulting in a distinct gradient of bands separated based on speed of travel through the matrix and subsequently size.
 * 2) The resulting bands of proteins are transferred onto a polymer sheet (nitrocellulose filter or polyvinylidene difluoride, PVDF) that binds proteins. This allows the proteins to bind to antibodies.
 * 3) Since the polymer sheet binds all proteins, the remaining empty space on the sheet must be blocked so it does not react with the antibody. This is done by washing the polymer sheet in non-fat dry milk, which contains casein (a protein), which will cover the rest of the sheet.
 * 4) The sheet is then incubated with the primary antibody specific for the target protein. After washed and diluted, the sheet is again incubated with the secondary antibody, which binds to the primary antibody. Usually, this secondary antibody is fuorescent or radioactive labeled. The sheet is again washed and diluted.
 * 5) The sheet is incubated with the enzyme substrate that would make the secondary antibody to emit light. Photographic films will detect this light as dark bands. The radioactive labeled antibodies are detected with X-ray films.



Factors Influencing Transfer

The buffer

A buffer that is commonly used for transfer is Tris-glycine which has pH 8.3 with a low concentration of SDS and 20% methanol. Glycine is used instead of Cl because it has a lower charge density and less current means less heat production. At this pH, many proteins will have a slightly negative charge which will make proteins transfer our easier since they will tend to move towards the positive charged electrode. SDS favors elution from the gel because it covers protein with negative charge. But if the protein is too negatively charged the hydrophobic membrane will repel the protein hence the protein will be less likely to transfer onto the membrane. To prevent that, methanol will be use to remove SDS from proteins which covers hydrophobic sites on the protein which can then bind to the membrane. If there is no methanol, elution from the gel is favored but binding to the membrane is poor. If there is too much methanol then the SDS could all come off before proteins leave the gel and the exposed hydrophobic regions could aggregate forming large protein aggregates that would be trapped in gel hence cannot transfer out.

The Membrane

There are different kinds of membrane with different pore sizes. The most commonly used membrane has pore sizes around 0.45 micrometer but smaller pore sizes are necessary to efficiently trap proteins that has size less than 20kDa.

Voltage during transfer

The current is more critical than the voltage. One of the factors here is the heating. The second factor is that if the current is too high, proteins with low molecular weight will move through the membrane so fast that they do not have a chance to bind with the membrane.

Time to transfer

The migration of the proteins will depend on the sizes. The larger the size of protein the longer time it will take to migrate. Sometimes two layers of membrane are placed next to the gel to trap smaller proteins that might pass through the first membrane.

Protein charge

If a protein has positively charged after SDS removed then they will be difficult to transfer. Since their isoelectric points can be similar to the pH of the buffer system. Hence, the protein will not migrate toward the positive electrode and not transfer onto the membrane. We can solve this problem by raising the buffer system to higher pH.

Advantages of Western Blotting
•Western blotting is effective and useful method to detect and characterize proteins in small amounts, such as clock proteins. Moreover, clock proteins’ other properties like half-life, molar amounts can also be found using western blotting.

•Immunogenic responses from infectious agents (ex. viruses, bacteria) are easy to detect by this technique.

•Western Blotting utilizes not only antigens, but also antisera as a diagnostic tool. Antisera is widely used in the test for HIV presence.

•Compared to ELISA, Western blotting has higher specificity; the higher specificity, the more the method is independent of the specificity of antibodies.

•Polyvinylidene difluoride (PVDF), or Nylon, is often used as membrane in Western blotting, since it has a high protein-binding capacity and chemical stability. Even, some protein groups only bind to Nylon or favor strongly to it.

•Among three common enzyme substrates, Fluorescent and Chemiluminescent create light detectable through X-ray or scanners. This ability enables high levels of sensitivity and quicker processing time.

Disadvantages of Western Blotting
•A non-intended protein has a slight chance of reacting with the secondary anti-body, resulting in the labeling of an incorrect protein.

•Incidental phosphorylation or oxidation of proteins may result in multiple bands appearing after sample is processed.

•The appearance of bubbles may occur when transferring the sample from the gel/membrane sandwich and may also occur when incubating the sample with antibodies, resulting in a skewed band reading.

•If the transfer time is not sufficient when transferring proteins to the membrane, the larger proteins of higher molecular weight will not transfer properly, resulting in an incorrect or no band reading at all.

•Too much methanol in the transfer buffer decreases the transfer efficiency of proteins from the gel to the membrane; however methanol aids in protein binding to several different membranes, so a correct balance is required.

•Western Blotting is a very delicate process requiring the correct amounts of each component in order for successful identification of the presence of proteins. An imbalance in any step of the procedure may skew the entire process.

Far-Western Blotting
Far- western blotting is based on the Western blotting technique. The major difference is that far-western  uses  non-antibody  proteins which  can  bind to the desired protein. Far-western is used to detect protein protein interactions.

Eastern Blotting
Eastern blotting is an extension of western blotting and is used in the detection of protein post-translational modifications. Eastern blotting is used to detect lipoylated, glycosylated or phosphorylated proteins or proteins with other post translational modifications. Blotting is mostly done from a SDS-PAGE gel on to a PVDF or nitrocellulose membrane. Post translational protein modifications including phosphorylation, lipoylation, glycosylation  or any other protein modifications  are detected  by specific probes.

Far-Eastern Blotting
Analyses lines separated by  high-performance thin layer chromatography (HPTLC). Further analysis is prepared by being transferred form the HPTLC plate to a PVDF then   by either performing enzymatic or ligand assays or mass spectrometry. A disadvantage to this procedure is the amount of downtime present in being able to prepare both a chromatography and a western blotting continuously since both require post experimental readings which have to be prepared separately.

Far-eastern blotting allows for the following techniques: ■	Purification of glycosphingolipids and phospholipids. ■	Structural analysis of lipids in conjunction with direct mass spectrometry. ■	Binding study using various ligands such as antibodies, lectins, bacterium, viruses, and toxins, and ■	Enzyme reaction on membranes.

Additional information
• In addition, it is important to know that in most cases, the blot is a nitrocellulose or polyvinylidene membrane, which is mobile when submersed in gel and electrical current is applied. In order for the proteins to move along the gel, the proteins must be soluble. A lysis buffer is needed to accomplish this. (Reference: www.abscam.com/ps/pdf/protocols/WB-beginner.pdf).

• Western Blotting is used in the human immunodeficiency virus (HIV) test. A blot is used to detect anti-HIV antibody in a human blood sample. The cells that have been infected by HIV have their proteins separated and blotted on the membrane. The human blood sample is then applied in the antibody incubation step and the free antibody is washed away, followed by a secondary anti-human antibody is added. After x-ray analysis, the bands that are stained show which proteins the person's blood contains antibodies to.