Fab Antibody Coupling to hIgM-FcCatchers Protocol
This protocol provides the steps to follow to couple a recombinant Fab antibody incorporating a SpyTag2 (Fab), e.g., format Fab-F-Spy2-H, to a hIgM-FcSpyCatcher3 (TZC036).
These guidelines can also be applied to couple a protein with a reactive SpyTag to a hIgM-FcCatcher. hIgM-FcCatcher is a disulfide-bridged dimer, and 5 or 6 of these dimers form a disulfide-bridged complex of the final hIgM-FcCatcher containing 10–12 SpyCatcher sites. The exact oligomeric state of the hIgM-FcCatcher is not used in the calculations for coupling; instead, these are based on the molecular weight of the dimeric protein.
Short Protocol
1. Calculate the required volumes of Fab and IgM-FcCatcher, starting with the amount of Fab you want to couple (see the online SpyCatcher-Coupling Calculator) or the detailed protocol below for calculations.
2. Mix Fab and IgM-FcCatcher.
3. Incubate for 1 hr at room temperature (RT).
Detailed Method
1. To ensure complete coupling with the IgM-FcCatcher, it is recommended to start with a 25% molar excess of Fab over SpyCatcher sites. Since each IgM-FcCatcher dimer contains two SpyCatcher sites, this corresponds to the 2.5-fold amount of Fab, e.g., 1 nmol hIgM-FcSpyCatcher3 + 2.5 nmol Fab-F-Spy2-H. It is possible to use a 2:1 coupling ratio for Fab:FcCatcher (equivalent to a 1:1 ratio for Fab:SpyCatcher sites), but inaccuracies in protein concentration determination and pipetting might lead to deviations from this ratio and to unpredictable amounts of uncoupled Fab or IgM-FcCatcher.
2. When coupling Fab and IgM-FcCatcher in solution, it is recommended to use the IgM-FcCatcher at the original concentration supplied, and to adjust the concentration of the Fab to 1 mg/mL if practical (Notes 1 and 2).
3. Add the required volume of IgM-FcCatcher to the Fab. Mix and incubate for 1 hr at RT. It is not important to stop the reaction after 1 hr, it can be left overnight if desired (Notes 3 and 4).
To calculate the amount of Fab and IgM-FcCatcher required make use of our SpyCatcher-Coupling Calculator for planning experiments.
Alternatively, assuming the IgM-FcCatcher is at the original concentration, if the Fab concentration is 1 mg/mL, the volume of IgM-FcCatcher required is 1/5 the volume of Fab, i.e., add 20 µL IgM-FcCatcher to 100 µL of Fab; if the Fab concentration is 0.5 mg/mL, the volume of IgM-FcCatcher required is 1/10 the volume of Fab, i.e., add 10 µL of IgM-FcCatcher to 100 µL of Fab.
To calculate the required volume of IgM-FcCatcher when starting with quantities or concentrations different from above:
| V(Fab): | Volume of Fab (µL) |
| m(Fab): | Amount of Fab (µg) |
| conc(Fab): | Concentration of Fab (mg/mL) |
| V(FcCatcher): | Volume of IgM-FcCatcher (µL) |
| Mw(Fab): | Molecular weight of Fab (g/mol) |
| c(FcCatcher): | Molar concentration of IgM-FcCatcher (µM) |
| Valency: | Number of Catcher sites, 2 per IgM-FcCatcher |
| Ratio: | Ratio of Fab:Catcher; 1.25 is recommended for the IgM-FcCatcher dimer |
Note 1. There is no minimum concentration required for coupling, but the coupling reaction is faster when the components are at a higher concentration; the lower the concentration, the slower the reaction.
Note 2. When working with dilute Fab or IgM-FcCatcher, e.g., immobilized IgM-FcCatcher on a resin or ELISA plate, and antibody concentrations in the single- or double-digit µg/mL range, reaction times for complete coupling will be longer and must be determined experimentally.
Note 3. This method can be used for coupling IgM-FcCatchers to SpyTag1, SpyTag2, and SpyTag3. A longer reaction time is required when coupling to SpyTag1.
Note 4. When an excess of SpyCatcher is used, there will be uncoupled SpyCatcher remaining after completion of the protein ligation reaction. It is recommended to block the SpyCatcher site by the addition of a 2.5-fold molar excess of SpyTag3 Peptide (catalog BLP086) over SpyCatcher, followed by incubation for 5 minutes at RT. This step is especially recommended for assays that contain more than one SpyTagged antibody, e.g., sandwich ELISAs or multiplex assays.
Recommended Storage
The success of the reaction can be checked using reducing SDS-PAGE with Coomassie staining. Run 1–1.5 µg of the coupled product. For comparison, also run the uncoupled Fab and IgM-FcCatcher on the same gel.
Quality Control
For short-term use, store aliquots at 2–8 °C; for long-term storage, refer to the conditions recommended on the datasheet for each specific FcCatcher. Avoid repeated freeze-thaw cycles. The addition of 0.01% 2-Methyl-4-isothiazolin-3-one (MIT) as a preservative is recommended for storage for up to one month at 2–8 °C.
Calculating the Molar Concentration of the Coupled Antibody
| c(Product): | Molar concentration of coupled antibody (µM) |
| n(FcCatcher): | Molar amount of IgM-FcCatcher used for the reaction (pmol) |
To convert the molar concentration to weight concentration:
| Mw(FcCatcher): | 102,448 Da |
| Mw(Fab): | ~ 50,700 g/mol |
Visit bio-rad-antibodies.com/spycatcher for further information.