Surelight apc anti 6x his tag antibody

Library sorting was facilitated by growing diploid library cells overnight in SD-Trp-Leu medium at 30°C and 120 rpm agitation. Subsequently, library cells were transferred to SG-Trp-Leu medium supplemented with 10% (w/v) polyethylene glycol 8000 at an OD₆₀₀ of 1.0 and incubated for 2 days at 20°C and 120 rpm. Afterwards, cells were washed twice with PBS (Sigma Aldrich) and incubated with C-terminally hexahistidine tagged recombinant human NKp30 ECD (produced in-house or Abcam) at a concentration of 1 µM for 30 min on ice. Cells were washed thrice, followed by simultaneous detection of functional Fab display and antigen binding. To this end, cells were labeled with light chain specific goat F(ab’)2 anti-human lambda R-phycoerythrin (R-PE) (SouthernBiotech, diluted 1:20) as well as Penta-His Alexa Fluor 647 Conjugate antibody (Qiagen, diluted 1:20) for sorting round one or SureLight^® APC Anti-6X His tag^® antibody (abcam, diluted 1:20) for sorting round two. Eventually, library cells were washed thrice with PBS and selected by fluorescence-activated cell sorting (FACS) on a BD FACSAria™ Fusion cell sorter (BD Biosciences). In the first round of selection, a total number of approx. 5 x 10⁸ cells were sorted to ensure adequate coverage of the library. For the second round of library sorting about 5 x 10⁷ cells were exploited.

For library sorting, cells were grown overnight in SD-Trp-Leu medium at 30°C and 120 rpm. Next day, cells were harvested by centrifugation and used to inoculate SG-Trp-Leu medium at an OD₆₀₀ of 1.0 and incubated for 2 days at 20°C. Fab expression was detected by incubation with light chain specific goat F(ab′)2 antihuman lambda R-phycoerythrin (R-PE) (SouthernBiotech, diluted 1:20). Antigen staining was conducted utilizing Penta-His Alexa Fluor 647 conjugate antibody (Qiagen, diluted 1:20) for sorting round 1 or SureLight^® APC Anti-6X His tag^® antibody (Abcam, diluted 1:20) for sorting round 2. For this, cells were harvested and washed twice with PBS (Sigma Aldrich) prior to incubation with EGFR at a concentration of 1 µM for 30 min on ice. After washing thrice, cells were then incubated with secondary labeling reagents for another 30 min on ice. Finally, cells were washed thrice with PBS and resuspended in appropriate volume for FACS sorting on a BD FACSAria™ Fusion cell sorter (BD Biosciences).