Provided with this kit is an NCX Blocking Buffer with a formulation of 4 mM sodium borate 1% BSA at pH 8.2 (pH 8.2 allows for optimum binding of BSA to antibodies). Blocking a conjugate can often decrease non-specific binding via the addition of non-specific proteins such as bovine serum albumin (BSA) or casein protein. NanoComposix standard protocols include a 1 hour blocking step with the supplied blocking buffer. Blocking is typically performed immediately following the quench step but can be done at any time post-conjugation.
- Aliquot 0.5 mL of the optimal conjugate from Experiment #4.
- Spin conjugate at 2k RCF for 5 minutes.
- Remove ~450 µL of supernatant and resuspend with ~450 µL NCX Blocking Buffer (4 mM sodium tetraborate, 1% BSA, 0.05% sodium azide, pH 8.2)
- Incubate/mix for 1 hour.
- Spin conjugate at 2k RCF for 5 minutes
- Remove supernatant
- Re-suspend in the NCX Conjugate Diluent (0.5× PBS, 0.5% BSA, 0.5% casein, 1% Tween 20, 0.05 % azide pH 8)
Compare performance of blocked conjugate to unblocked conjugate. If the blocking step improves performance, it is recommended to investigate the following blocking times: 30 minutes, 1 hour, 2 hour, and overnight. If the blocking step did not demonstrate an improvement in conjugate performance or stability, we recommend that you do not include a blocking step.