When performing Western blots it is essential that the amount of both primary and secondary antibody is optimised to reduce background and ensure that adequate signal is achieved. Our gyrase polyclonal and monoclonal antibodies have already been tested to reduce this need for further optimisation. *
The primary antibody should be diluted 1:1,000 where pure protein has been loaded or 1:200 for cell lysates. Dilutions are usually made in wash buffer (1X TBS, 0.1% Tween 20, 1% dried skimmed milk), 20 ml per blot, and incubated for 1 hour. After washing with wash buffer to remove the primary antibody, the secondary antibody is added at 1:5,000 dilution in wash buffer (anti-rabbit for polyclonal, anti-mouse for monoclonal).
Blots can then be visualised using standard colorimetric or chemiluminescent methods.
* Optimisation conditions were for SDS-PAGE mini-gels where 1 µg of protein sample (purified protein or cell extract) has been loaded in each lane and gels run under standard SDS-PAGE conditions before blotting (200 mA for 45 mins for 43 kDa protein or 60 mins for 90 kDa or 97 kDa proteins).