Commercially available protein labeling kits provide researchers with the flexibility to customize their own immunoassay detection panels. However, because many protocols require buffer exchange prior to and following antibody labeling, dialysis-based workflows are time-consuming and subject to significant protein loss at multiple points of sample transfer.
For removing unincorporated label in protein labeling applications, centrifugal devices are a fast, convenient, high-recovery alternative to gel filtration.
Significantly higher yields can be achieved from antibody labeling by combining the labeling step with the clean-up step in the all-in-one Amicon® Pro system. The Amicon® Pro purification system is an adaptable centrifugal device coupling affinity purification with downstream sample concentration and buffer exchange.
Two attributes of the Amicon® Pro device make it a convenient device for preparing pure, labeled antibody:
For details on the Amicon® Pro Antibody Labeling application, please visit our dedicated Antibody Labeling page, as well as our publication (Read our 2015 paper in Journal of Immunological Methods)
Read below for a brief summary of the protocol for using the Amicon® Pro device for small–scale antibody labeling.
NOTE: All steps, with the exception of binding reactions, are spin-based. A swinging bucket rotor is required for all steps with the exception of the reverse spin.
Optional: Pre-wet device using 0.5 mL TBS-T. Spin at 1000 x g for 1 min.
To compare the functional performance of antibodies labeled using traditional centrifugal diafiltration and using the Amicon® Pro method, their detection ability was compared with that of a species/anti-species GAPDH detection pair. Using similar amounts of primary antibody for each blot, both biotin/SA pairs demonstrated the same, if not slightly better sensitivity, than the species/anti-species detection pair (Figure 1).
Anti-GAPDH, biotinylated using the Amicon® Pro system, was used to successfully detect GAPDH in Western blots. Aliquots of EGF-stimulated A431 cell lysate (0.25-4 µg) were resolved by SDS-PAGE, transferred onto Immobilon®-P membrane, and probed for β tubulin in the SNAP i.d.® 2.0 system using the various indirect detection pairs. In each case, the same quantity of primary (GAPDH-specific) antibody was used.
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