RPSA | 30S ribosomal protein S1 (bacterial)

6 µg/well of total protein extracted freshly from E. coli strains MG1655, 536, UTI89, RS218, IHE3034 and IHE3034ΔycgG2 with 20mM Tris buffer and extracted by freeze-thawing (3 cycles). The samples were then centrifuged for 30 min at 13 000 rpm and the protein content of the supernatants was subject to measuring with the BCA method. The proteins were separated on an 10 % SDS-PAGE and blotted onto a PVDF (pore size of 0,42 µm) membrane, using semi-dry transfer. The membrane was blocked with 5 % milk at 4°C/ON with agitation, followed by washing in PBS-T buffer (3 times for 15 min) before it was incubated with the primary antibody at a dilution of 1: 3 000 in PBS-T for 1h/RT with agitation. After that, the membrane was washed once for 30 min and 2 times for 15 min in PBST at RT with agitation. Then it was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated AS09 602) diluted 1:30 000 in PBS-T for 1h/RT with agitation. Finally, the immunoblot membrane was washed for 15 minutes three times with PBS-T and developed for 5 min with Agrisera ECLBright before imaging with a LAS 4000 image reader. Exposure time was 30 seconds.

Courtesy of Moa Näsman/the Uhlin's Lab, Umeå University, Sweden