Alexa fluor 488 microscale protein labeling kit

Purified lipoarabinomannan (LAM) from Mtb H37Rv and Mycobacterium smegmatis were obtained through BEI Resources, NIAID, NIH, USA. LAMs were labeled with the Alexa Fluor 488^® Microscale Protein Labeling Kit (life technologies). A constant amount of Alexa488-labeled LAM (200 nM) was incubated for 15 min at 37 °C with serially diluted concentrations of IL-26, LL37 or IL-22 (R&D Systems) in microscale thermophoresis (MST) binding buffer (50 mM Tris and 0.05% Tween 20). Ten μL of the samles were loaded into standard Monolith NT Capillaries and analyzed on a NanoTemper Monolith NT.115 apparatus.

The native polyacrylamide gels used for Redβ and DNA band-shift assays were cast according to standard procedures. For a final volume of the separating gel of 50 ml (20 ml of the stacking portion) the following components were mixed: 40% Acrylamide (19:1 Bis.) 10 ml (2.5 ml), 10x TBE 12.5 ml (2.5 ml), H₂O 27 ml (14.8 ml), TEMED 30 μl (20 μl), 10% APS 500 μl (200 μl). The samples contained 25 fmol of 30-b-long Atto 565-ssDNA_B (Table B in S1 Text) and Redβ with a constant 5 nM chemically labelled with Alexa 488 using the Alexa Fluor 488 Microscale Protein Labeling Kit (Invitrogen). Prior to application on the gel, the ssDNA was incubated with Redβ for 20 min. For filament formation, 1 nM of a 30-b-long 5′ Atto 565-labelled and 0.67 nM of a 50-b-long ssDNA were incubated for 20 min with Redβ and united, followed by 15 min of incubation, producing a 5′ overhang. A saturated sucrose solution was mixed with the sample in a ratio of 1:6 (vol/vol) to increase its density for loading. The samples were applied to a 5% stacking and 8% separating gel and run for 120 min at 100 V at 4°C in 1 × TBE buffer. The gel pictures were recorded with a Typhoon 9410 Imager. To visualize the bands of Fig 2 using ethidium bromide staining, we used a ssDNA concentration of 50 μM. Accordingly, we used a high concentration of unlabeled Redβ of 100 μM.