A simple yet effective detection method
Dot blots are very similar to Western blots in that they involve the use of antibodies to identify a protein that has been bound to a membrane. They do not however require electrophoretic protein separation on a gel; the test sample is simply spotted on to the membrane and then detected.
Due to the lack of a protein separation step, dot blots cannot be used to determine the molecular weight of a protein or to discriminate between different protein forms (e.g. cleaved or phosphorylated proteins). Instead dot blots are convenient for estimating the protein concentration in crude preparations such as tissue culture supernatant or ascites, for determining whether an antibody-based detection system will work effectively, or to identify an appropriate antibody concentration for Western blotting.
The main steps of a traditional Dot Blot assay are as follows:
- The protein is spotted on to a nitrocellulose or PVDF membrane, for example using a low-volume pipette or a pin tool
- The membrane is blocked
- Incubation with primary antibody
- The membrane is washed to remove unbound antibody
- Incubation with secondary antibody
- The membrane is washed to remove unbound antibody
- Detection – usually colorimetric, fluorometric, chemiluminescent or using gold nanoparticles
The use of directly-labeled antibodies removes the need to carry out steps 5 and 6, significantly decreasing assay time and potentially reducing non-specific binding and any associated background staining. Direct antibody labeling is discussed in further detail at the end of this application note.
Use of dot blots to estimate protein concentration
Through spotting a purified protein in a series of known concentrations on to a membrane, a dot blot can be used to estimate the concentration of a target protein in a test sample. The concentration of protein in the test sample can be extrapolated from the serial dilution of purified protein.
Figure 1. The left hand side of the membrane shows a serial dilution of purified protein, while the right hand side contains three different dilutions of the test sample. The serial dilution can be used to estimate the concentration of protein in the test sample.
Use of dot blots to evaluate an antibody-based detection system
A dot blot can be used to establish whether an antibody-based detection system will work effectively, and if it therefore has the potential to be converted to a Western blot approach. After spotting purified protein and test sample on to the membrane, the membrane is incubated with appropriate primary and secondary antibodies to determine whether a signal can be detected. The primary antibody can also be spotted on to the membrane to ensure that the secondary antibody is compatible with it.
Figure 2. The membrane is spotted with three different dilutions of recombinant protein, test sample and primary antibody. The protein can be detected with primary and secondary antibody; the primary antibody dots can be detected with secondary antibody only.
Use of dot blots to identify an appropriate antibody concentration
The best Western blot results are obtained when the detection antibodies are used at their optimal concentrations. One way of determining these concentrations is to produce a dot blot that has been spotted in a grid pattern with a known concentration of protein. Through spotting different concentrations of primary antibody across the blot, and different concentrations of secondary antibody down the blot, the optimal staining conditions can easily be established.
Figure 3. The membrane is spotted in a grid pattern with a known concentration of protein. The protein is then detected with a range of dilutions of the primary and secondary antibodies. An appropriate combination of antibody concentrations should give rise to a level of signal that is not at the lower end of the detection limits (top left) or so high that the signal burns out (bottom right). It can be beneficial to include no primary or no secondary antibody controls to identify the source of any background staining.
It is often beneficial to use a directly-labeled primary antibody, since this bypasses the need for a secondary antibody incubation step.
The benefits of the direct approach are as follows:
- Non-specific binding is avoided since secondary antibodies are not used
- Multiplexing is possible with antibodies from the same species
- Faster since there is no secondary antibody incubation step and therefore fewer wash steps
- Data quality is improved through assay simplification
Lightning-Link® from Expedeon is an innovative technology that enables direct labeling of antibodies with enzymes, fluorophores, biotin or streptavidin. Labeling antibodies with enzymes permits colorimetric or chemiluminescent detection to be performed, while fluorometric detection provides the option of multiplexing.
The benefits of Lightning-Link® include:
- It is quick and easy to use
- It requires only 30 seconds hands-on time
- There are no separation steps involved so you recover 100% of your antibody or protein
- You can label from as little as 10ug to a gram or more!
As an alternative to using enzymes or fluorophores for detection, antibodies can instead be directly conjugated to gold nanoparticles. Binding of the gold-conjugated antibody to its immobilized protein target gives rise to a red color on the membrane, and this signal can be boosted through silver enhancement if necessary. InnovaCoat® GOLD enables direct labeling of antibodies with gold nanoparticles that have a proprietary surface coating. The benefits of InnovaCoat® GOLD include:
- Antibodies can be covalently linked to gold nanoparticles in just 15 minutes
- Metal-antibody interactions are prevented
- No pH titrations are required
- Different surface chemistries are available
A directly conjugated primary antibody can easily be used for the three applications discussed here. Identification of an appropriate antibody concentration is simplified since the secondary antibody no longer needs to be taken in to account.Click to read more