Laemmli sds loading dye

Overnight cultures were diluted 1:100 in fresh LB medium and incubated at 37 °C with shaking (250 rpm) until an OD₆₀₀ of 0.4 was reached. Cultures were infected with DMS3^6HisTT at an MOI of two. At timepoints 0, 30, and 60-min post-infection, 5 mL of cells were collected by centrifugation and flash frozen at −80 °C. The bacterial pellets were resuspended in 100 μL ice-cold sonication lysis buffer and were sonicated at 30 kHz for 5 min (30 s on, 30 s off). Cleared cell lysates were mixed with 2X Laemmli SDS loading dye (Bio-Rad), boiled at 100 °C for 20 min and then separated by 15% SDS-PAGE. For Western blot analyses, proteins on SDS-PAGE gels were transferred to a 0.45 μm nitrocellulose membrane using Bio-Rad Trans-lot SD Semi-Dry Transfer Cell at 15 V for 1 h. Western blots were incubated with primary mouse anti-His₆ monoclonal antibody (BioShop TAG001.100; diluted with TBS-T buffer containing Bovine serum albumin at 1:5,000) at 4 °C overnight, and then with secondary anti-Mouse IgG Horseradish peroxidase-linked antibody (Cell Signalling Technology; diluted with TBS-T buffer containing Bovine serum albumin at 1:10,000). Western blots were developed using Bio-Rad Clarity^TM Western ECL substrate (Cat. # 170-5060) and imaged with the Bio-Rad ChemiDoc Imaging system.

His₆-tagged Tab was cloned alone (using primers PHP160-PHP161) or in combination with untagged Anti-Tab (using primers PHP162-PHP163) into the pET-Duet-1 vector for protein co-expression and purification. These plasmids were transformed into E. coli BL21(λDE3) for protein expression. Overnight cultures were diluted 100-fold into fresh LB and grown to an OD₆₀₀ of 0.8. Protein expression was induced with 1 mM of IPTG, and the cultures were grown for three hours. Following the induction period, the cells were collected by centrifugation, the cell pellets were resuspended in binding buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, and 5 mM imidazole) and the cells lysed by sonication at 30 kHz for 5 min (30 s on, 30 s off) on ice. Cell debris was collected by centrifugation at 21,000 xg for 20 min at 4 °C. The insoluble fraction was resuspended in fresh binding buffer. The protein samples were mixed with 2X Laemmli SDS loading dye (Bio-Rad), boiled at 100 °C for 10 min and then separated on 15% SDS-PAGE gels before staining with Coomassie blue and subjecting to Western blot analysis as described above.