Fuji las 4000 imager

16 μl of varying concentrations of siRNA-binding peptides in water were added to 50 μl of PBS and incubated for 5 min. at 20^°C. In some experiments, the E9 peptide was added for 5 minutes. 5 μl of 20 μM siRNA solution was then added to each tube, mixed briefly on a vortex, and then incubated for 15 min at 20^°C. 5 μl was then removed from the mixture and added to a tube containing 1 μl of 6X non-denaturing agarose gel loading buffer containing 30% glycerol in water, 0.25% bromophenol blue and 0.25% xylene cyanol FF. The contents were mixed, and then 5 μl loaded onto a 2.5% agarose gel in TBE buffer (0.09 M Tris, 0.09 M boric acid, and 0.002 M EDTA). Following electrophoresis to resolve the mixture, the gel was stained with Sybr Green II RNA stain (In Vitrogen) for 30 minutes and the fluorescent bands visualized using a Fuji LAS4000 Imager.

Native gels were scanned with a Typhoon Trio (GE Healthcare) scanner in fluorescence acquisition mode; excitation: 488 nm, emission: 526 nm.
Western blotting was done as follows: first, the native gels are pretreated by immersion in a solution containing 1% (w/v) dithiothreitol (DTT) and 2% (w/v) sodium-dodecylsulfate (SDS) at RT for 15 minutes. The treatment breaks the in-gel protein complexes and facilitates the transfer of proteins from the gel to the PVDF-membrane, which was subsequently done by semi-dry Western blotting. The blots were probed with antibodies against a series of cell division proteins α-FtsQ, α-FtsK, α-FtsN (kind gifts from the De Gier, Weiss, Beckwith, Vicente and Den Blaauwen labs), α-FLAG (Sigma-Aldrich) and control proteins (α-SecY, α-F_oc: own collection). Subsequently, immunodetection was done using alkaline phosphatase conjugated anti-rabbit IgG, F(ab’)₂fragment (Sigma) and CDP-star (Roche). Chemiluminescence was detected using a Fuji LAS-4000 imager. Whole cell samples from the same material were run on SDS-PAGE gels, Western blotted, and probed to compare total protein levels in the cell samples. In the case of FtsQ wt and FtsQ D237N overexpression, the relative quantity of the probed proteins (thickness of bands) was analyzed with FIJI59 (link).