The Dot Blot
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 Innova Biosciences 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
Lightning-Link® from Innova Biosciences is an innovative technology that enables direct labeling of antibodies, proteins, peptides or any other biomolecule which has free amine groups. These easy to use kits require just 30 seconds hands-on time and, with no separation steps involved, 100% of the biomolecule is recovered. Lightning-Link® conjugates are highly stable, and with…View
Download a Free Copy Lightning-Link® fluorescent antibody and protein labeling kits allow you to covalently label any antibody in under 20 minutes with only 30 seconds hands-on time. We currently have over 35 label in our range of fluorescent dyes. This guide provides a general overview of each fluorochrome in our range including…View
The affinity of streptavidin for biotin is the strongest non-covalent biological interaction known, with a dissociation constant (Kd) in the femtomolar range. Each streptavidin monomer can bind one biotin molecule, allowing a streptavidin protein to maximally bind four biotins. The streptavidin-biotin interaction In addition to the strength of the interaction, the binding of streptavidin to…View
Antibodies are widely employed in the quantification of antigens in complex biological samples. Using techniques such as Western blotting, ELISA, and immunohistochemistry researchers are able to measure a single antigen, or perhaps a limited number of antigens, in each sample. In the post-genomics era, advances in multiplex immunoassay technologies now allow scores or even hundreds…View
Innova Biosciences specializes in easy to use bioconjugation kits which enable the direct labeling of antibodies or proteins with enzymes, fluorescent labels, biotin, streptavidin, gold nanoparticles, latex beads or oligonucleotides. Unfortunately, many antibodies are provided in buffers which contain additives that are incompatible with labeling technologies, making puri cation a key consideration prior to carrying…View
Antibodies are used to detect and quantify antigens in techniques such as flow cytometry, ELISA, western blotting, immunohistochemistry and lateral flow. The antibody that binds to the antigen is called a ‘primary antibody’ and it confers specificity to the assay. Most types of immunoassays also incorporate a ‘label’ whose purpose is to provide measurability. Assays…View
Stop using secondary antibodies in immunoassays! Save time and materials by labeling your primary antibodies in under 20 min. For use in ELISA, western blotting, IHC, flow cytometry and many other applications. Scientists love our Lightning-Link® kits because of the remarkable simplicity of the technology. Now they can open a…View
Dr Nick Gee, the CEO and CSO of Innova Biosciences, explains the benefits of using directly conjugated antibodies and the various approaches and techniques that researchers can use in their own labs. The webinar covers: Why conjugate your antibody? What are the main chemical approaches? Is there a 'best' method?…View