Secondary Antibody Frequently Asked Questions

Secondary antibodies are raised against the species of the primary antibody. Therefore, you need a secondary antibody raised in a species different from the host species of your primary antibody. For example, if your primary antibody was raised in rabbit, you will need an anti-rabbit secondary antibody raised in a non-rabbit host species (e.g., goat, mouse, etc.).

Some of our recommended antibody dilution/concentration values are derived from experiments during antibody development or based on the experience of our collaborators and other customers. Due to differences in samples or experimental conditions, they may not work optimally in your specific experiment. If our recommendation does not work for you, please try testing several different dilutions/concentrations.

Goat Anti-Mouse IgG (H+L) is available in several grades, and there is some sequence similarity between IgG and IgM antibodies. Therefore, Goat Anti-Mouse IgG (H+L) that has not been cross-adsorbed may exhibit some degree of cross-reactivity with mouse IgM antibodies. If complete absence of cross-reactivity with IgM antibodies is required, you can choose antibodies such as Goat Anti-Mouse IgG1, Goat Anti-Mouse IgG2a, Goat Anti-Mouse IgG2b, etc.

Goat Anti-Mouse IgG (H+L) will bind to all subclasses of mouse IgG, including IgG3.

This antibody has not been cross-adsorbed against rat IgG, and we do not have extensive information on this. However, considering that the antibody in question is polyclonal, this possibility cannot be ruled out.

The immunogen for Goat Anti-Mouse IgG (H+L) includes both heavy and light chains. The light chain can be either kappa or lambda. Therefore, this antibody will react with primary antibodies carrying kappa chains, but it is not specific solely for the kappa chain.

An antibody labeled with two different conjugates enables both chromogenic and fluorescence imaging on the same sample. Therefore, multimodal studies are possible, and we can provide custom labeling for this purpose.

Secondary antibodies bind to primary antibodies. Therefore, the application range of a secondary antibody is more closely related to the primary antibody. If a primary antibody can be used in certain immunoassays, the secondary antibody can typically be used as well. Consequently, the final application scope depends primarily on the application range of the primary antibody, the type of secondary antibody label, and the signal output system.

All fluorophore will fade or undergo "photobleaching" to some extent when exposed to intense light at their absorption wavelength. Potential solutions include:

1. Choose more photostable dyes, such as Fluor dyes.
2. Flexibly set the counterstain and field of view, then switch to the target stain for imaging.
3. Reduce exposure time and intensity, for example by lowering laser power or using neutral density filters.
4. Keep the observation time of labeled samples as short as possible, and close the shutter when not viewing.
5. Use objectives with lower numerical aperture, such as low-power objectives.

The classic ELISA system often uses a sandwich assay format, involving a capture antibody and a detection antibody. If both antibodies are mouse monoclonal antibodies, the HRP-labeled Goat Anti-Mouse IgG would bind to both, rendering the assay non-specific. Therefore, in an ELISA system, the host species of the capture antibody must not be mouse, while the detection antibody must be mouse-derived. Additionally, the sample type being detected should not contain mouse IgG or similar molecules.

Using secondary antibodies conjugated with different fluorophore for multicolor immunofluorescence is achievable, but requires careful design and consideration:

1. Fluorophore Selection: Choose fluorophores whose excitation and emission spectra are as separated as possible. Less spectral overlap reduces the possibility of crosstalk (bleed-through). Prefer dye combinations with widely spaced emission peaks (e.g., the combination of Fluor488, Fluor555, and Fluor647 is very classic).
2. Instrument Compatibility: Ensure that the fluorophore you select can be effectively excited and detected by the lasers and filter sets of your laboratory's microscope or imaging system. Consult the instrument configuration manual before selecting dyes.
3. Matching Brightness and Expression Level: Assign the brightest fluorophores (e.g., Fluor647, Cy5) to low-abundance target proteins. Assign dimmer fluorophores (e.g., FITC, Cy2) to high-abundance target proteins. This helps balance signal intensities across channels, preventing strong signals from masking weak ones.
4. Secondary Antibody Specificity and Cross-Adsorption: This is crucial for avoiding non-specific cross-reactivity and directly impacts result reliability. You must select secondary antibodies that have been cross-adsorbed. This means the antibody has been purified to remove antibodies that might cross-react with immunoglobulins from other species. For example, if you are using both mouse and rat primary antibodies, your anti-mouse secondary antibody should be cross-adsorbed against rat serum proteins, and vice versa. This ensures the secondary antibody only binds to its specific targeted primary antibody and does not cross-react with primary antibodies from other species.
5. Host Species: All secondary antibodies should be derived from the same host species (e.g., all goat-derived). This prevents secondary antibodies from recognizing and binding to each other (e.g., an anti-rabbit secondary accidentally binding to an anti-mouse secondary), which would cause false positives.
6. Experimental Design and Controls: Without proper controls, multicolor experiment results can be difficult to interpret. Single-stain controls are essential. For each experiment, include samples stained with only one primary/secondary antibody pair, while other channels receive only secondary antibody diluent (or no primary). Use these controls to check for crosstalk in each channel under your actual image acquisition settings. Blank Control: Omit all primary antibodies, incubate only with the secondary antibody mixture, to detect non-specific binding of the secondaries. Sequential Incubation: It is recommended to incubate primary antibodies from different species separately first, then incubate the corresponding species-specific secondary antibodies as a mixture. This can reduce the risk of cross-reaction compared to mixing all primaries and then all secondaries.
7. Antibody Concentration Optimization (Titration): Always titrate each antibody pair before use to find the optimal concentration that gives the highest signal-to-noise ratio. Excessively high antibody concentrations increase background and non-specific binding.
8. Image Acquisition and Processing: Even with a well-executed experiment, improper image acquisition can ruin the results. Avoid simultaneous excitation and acquisition of all channels. Set up the acquisition sequence to excite and capture each channel separately (sequentially). This completely eliminates crosstalk between channels during acquisition. Spectral Unmixing: If your instrument has this capability (e.g., some confocal microscopes), it can effectively separate fluorescent signals with highly overlapping spectra and is a powerful tool for multicolor experiments. Post-processing: If minor crosstalk is detected, software can sometimes be used for spectral unmixing calculations, but the best approach is to avoid crosstalk during the experimental design phase.