Primary anti gfp

Mixed-stage populations of worms were harvested, washed three times in M9 buffer, and the pellets were lysed in 2x Laemmli buffer. For analysis of the additional candidates (Fig 4) 60–80 L4 stage animals were picked for western blotting. For analysis of endogenous HSP-6, 100 L4 larvae were harvested per genotype. The protein extracts were separated by 10% SDS-PAGE and transferred to a PVDF membrane (0.45 μm pore, Merck Millipore). To detect GFP and Tubulin, we used primary anti-GFP (1:1000, Roche 11814460001) and primary anti-α-Tubulin (1:5000, Abcam ab7291) antibodies and secondary horseradish peroxidase-conjugated goat anti-mouse antibodies (BioRad #1706516). To detect endogenous HSP-6, we used anti-HSP-6 (1:10,000) as described previously [42 (link)] and secondary horseradish peroxidase-conjugated goat anti-rabbit antibodies (BioRad #1706515). Blots were developed using ECL (Amersham) or ECL Prime (Amersham) according to manufacturer’s protocol and images were quantified using the ChemiDoc XRS+ System (BioRad).

~10⁸ bacteria were harvested from appropriate cultures by spinning down cells equivalent to OD₆₀₀ = 1. Bacterial pellets were then resuspended in 1× SDS loading buffer (60 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue, 5 mm EDTA, 0.1 m DTT), boiled for 5 min and separated on a 4–12% Invitrogen NuPage novex Bis-Tris mini gels as detailed by Boysen et.al [87 (link)]. The proteins transferred onto PVDF membranes (Amersham Hybond) were hybridized with 1:20000 dilution of primary anti-GFP (Roche), 1:2000 anti-Fim, 1:1000 anti-PapA and a loading control of 1:50000 dilutions of primary anti-GroEL as appropriate. 1:2000 dilutions of appropriate secondary antibodies (Dako, Denmark) were used in the SNAP i.d.2.0 blotting system (Millipore). Chemiluminescent visualization of protein bands was performed using the Thermo Scientific Pierce ECL Western Blotting Substrate according to the manufacturer’s instructions.