- Proteins and Peptides
- Lysates and Cell Lines
What secondary antibody should I use with my primary?
This will vary depending on the primary antibody that you are using. The general rule is that you will want to select a secondary that matches both the host species and isotype of the primary antibody that you are using. For very specific isotypes (i.e. IgG2 or IgG1), a more general secondary antibodies can be used (i.e. IgG) depending on what you are detecting. For more complicated experiments, you should contact Technical Support for additional assistance in the selection process.
What dilution should I use for my application?
This will vary depending on the product. You can find the recommended dilution range under “Applications/Dilutions” section of the product datasheet (see below for an example).
When do I need to use the fragment specific secondary antibodies, such as Fc, F(ab) and F(ab’)2?
Fragment specific secondary antibodies can be used when the primary antibody you are trying to detect is comprised of only that specific fragment or when you are trying to only detect that specific fragment from a population of multiple immunoglobulin molecules. It can also be helpful when a particular antibody will have an Fc or F(ab) region exposed, due to the specific binding of that primary.
What is the difference between using a directly conjugated primary antibody versus a conjugated secondary?
Using a directly conjugated primary antibody in place of a standard primary/conjugated secondary set up will allow you to stream line your experiment and stain multiple targets on a single sample with little to no background interference. However, if the protein of interest is expressed low amounts in the sample being examined, it is more ideal to use a conjugated secondary. The conjugated secondary will provide some amplification of the signal as multiple secondary antibodies can bind to a single primary.
I am using a polymer based detection kit instead of a standard secondary antibody. I am getting some high background. Do you have a recommendation to help improve my staining?
A serum blocking step may not be required when using a polymer based secondary but if you are getting background issues, blocking with 5% goat serum may be employed. We would still recommend performing a peroxidase blocking step as well if the polymer is utilizing a peroxidase detection method.
What is the advantage of using the pre-adsorbed secondary antibody?
Using the secondary antibody that is pre-adsorbed against species of the sample being examined (i.e. mouse, rat, porcine, etc.) will minimize non-specific binding of the secondary antibody to the sample.
Should I use Fab specific secondary antibodies when using a mouse primary antibody on mouse tissue?
F(ab) specific secondary antibodies may help to reduce background signal by eliminating nonspecific binding of the IgG proteins found in tissues by the secondary antibody. You will want to make sure that the primary antibody you will be detecting will have an exposed F(ab) region for detection by the secondary. If not, you may want to consider using a Mouse on Mouse blocking reagent or an unconjugated anti-mouse IgG secondary prior to primary incubation as alternative methods for reduction of background signal.
Is it necessary to use a specific isotype/subclass of the secondary antibody when using a monoclonal primary antibody?
Usually it is not required to use the isotype specific secondary antibody with a monoclonal primary antibody. For most purposes, you can use a general immunoglobulin isotype (IgG, IgM, etc) to detect all subtypes of these isotypes. For example, you can use an anti-Mouse IgG secondary antibody to detect mouse IgG1 and IgG2 primary antibodies.
I have some leftovers of secondary antibody that has been diluted in 5% skim milk. How long can I store before it loses its activity?
The shelf life of prediluted secondary antibodies can vary greatly depending on the number of uses, storage temperature, age of storage buffers, and other handling factors. We recommend keeping the bulk of your secondary antibody in its original vial, only removing enough material for individual use. If you do decide to store prediluted secondary, you may need to conduct stability tests of the secondary antibody with your primaries to establish a working timeline for your needs.