Western Blotting Principle

Transfer

Proteins are moved from within the gel onto a membrane made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF). Without pre-activation, proteins combine with nitrocellulose membrane based on hydrophobic interaction, thereby having slight effect on protein activities. Besides, nitrocellulose membrane produces little non-specific staining. It is cheap and ease to use. However, it is easy to erase small molecular proteins while washing. It is fragile and has poor toughness. With high affinity, the PVDF membrane needs to be sunk in methanol before use to activate positive charge groups on the membrane, promoting combination with negative charged proteins. Specific NC membrane with different pores should be applied according to the molecular weight of transferred proteins due to the smaller the pore of membrane the tighter the combination between membrane and small molecular weight proteins. NC membranes of 0.45 µm and of 0.2 µm are used most. The size of 0.45 µm should be applied for proteins with molecular weight over 20KD while the size of 0.2 µm will be chosen for those below 20KD. PVDF membrane is best for the detection of small molecular weight proteins due to its higher sensitivity, resolution as well as affinity than normal membrane.

Transfer methods that are used most for proteins are semi-dry transfer and wet transfer. Semi-dry transfer describes the method that Gel-Membrane-Filter sandwich is placed between filters loaded with transfer buffer. The transfer process is based on current conduction produced by the transfer buffer. Semi-dry transfer takes little time with high efficiency as electric current works directly on membrane and gel. While applying wet transfer, the Gel-Membrane-Filter sandwich is placed in the transfer tank, suspending in transfer buffer vertically. Proteins transfer from the gel to the membrane under the control of high intensity electric field produced by electrode plate paralleled to the sandwich. While prolonging time to an appropriate extend, proteins could be transferred more effectively. Proteins within several gels could be transferred.

Blocking

In a western blot, it is important to block the unreacted sites on the membrane to reduce the amount of nonspecific binding of proteins during subsequent steps in the assay using inert protein or nonionic detergent. Blocking buffers should block all unreacted sites. And Blocking buffers should not replace target protein on the membrane, not bind epitope on the target protein and not cross react with antibody or detection reagents. The most typical blockers are BSA, nonfat dry milk, casein, gelatin and Tween-20. TBS and/or PBS are the most commonly used buffers.

Inertia protein BSA, nonfat dry milk, casein, gelatin or nonionic detergent Tween-20 reduce nonspecific binding by blocking unreacted sites. Retaining protein structure, Tween-20 can reduce breakup to original interaction among proteins while is used for protein emulsification.

1. Nonfat dry milk is the most economic choice
2. Avoid using nonfat dry milk as a blocking reagent for blots with biotin conjugated antibody because milk contains variable amounts of glycoprotein and biotin.
3. BSA is appropriate for blots with phosphorylated protein as target. Phosphatase contained in nonfat dry milk leads to dephosphorylation of phosphorylated protein on the membrane while phosphoryltion specific antibody is used to identify phosphorylated protein. And nonfat dry milk is improper for blots which rely on alkaline phosphatase system.
4. Avoid adding NaN3 into blocking reagent for blots that base on HRP system because NaN3 is enabled to inactivate HRP.
5. Casein is recommended for blots with alkaline phosphatase conjugated secondary antibody. TBS buffer instead of PBS buffer should be chosen because PBS interferes alkaline phosphatase.

Primary Antibody incubation

After blocking, primary antibody specific to target protein is incubated with the membrane. And the primary antibody binds to target protein on the membrane.

In western blot, primary antibody should be validated before use. The choice of a primary antibody depends on the antigen to be detected. Both polyclonal and monoclonal antibodies work well for western blot. Monoclonal antibodies recognize single specific antigenic epitope. Thus, they have higher specificity resulting in lower background. Blot results will be influenced if the target epitope is destroyed. Polyclonal antibodies recognize more epitopes and they often have higher affinity. Blot results will be stable even though a few epitopes are destroyed.

Secondary antibody incubation

After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to a specific enzyme-conjugated secondary antibody. And the secondary antibody binds to the primary antibody which has reacted with the target protein.

The most popular secondary antibodies are anti-mouse and anti-rabbit immune globulin since the host species for primary antibodies are mainly mouse and rabbit. Goat is used widely to raise anti-mouse and anti-rabbit polyclonal antibodies. Thus, goat anti-mouse and goat anti-rabbit immune globulin are the most commonly used secondary antibodies. The choice of secondary antibody depends upon the species of animal in which the primary antibody was raised. For example, if the primary antibody is a mouse monoclonal antibody, the secondary antibody must be an anti-mouse antibody. If the primary antibody is a rabbit polyclonal antibody, the secondary antibody must be an anti-rabbit antibody.

A substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance, namely, visible protein bands. The target protein levels in cells or tissues are evaluated through densitometry and the location of the visible protein bands.

Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes that are used extensively. Functioned by Alkaline phosphatase (AP) catalyzation, a colorless substrate BCIP will be converted to a blue product. In the presence of H2O2, 3-amino-9- ethyl carbazole and 4-chlorine naphthol will be oxidized into brown substance and blue products respectively under the catalyzation of HRP. Enhanced chemiluminescence is another method that employs HPR detection. Using HRP as the enzyme label, luminescent substance luminol will be oxidized by H2O2 and will luminesce. Moreover, enhancers in this substrate will enable a 1000-fold increase in light intensity. HRP will be detected when the blot is sensitized on photographic film.

Western Blotting Analysis

After color development, the pattern of the separated proteins is imprinted onto the film or captured by Western Blot gel imager. By comparing the band position to the protein ladder, one can estimate the size of the protein.