Ap conjugated rabbit anti human igg h l

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was performed using 12% mini-PROTEAN^® TGX™ precast gels (Bio Rad, Hemel Hempstead, UK). P. pastoris culture supernatants were denatured by boiling for 5 min in either reducing or non-reducing SDS sample buffer (0.0625 M Tris–HCl, pH 6.8, 2.3% (w/v) SDS, 10% (w/v) glycerol and 0.01% Bromophenol blue. ± 5% (v/v) β-mercaptoethanol). Molecular weight was estimated by using a prestained protein ladder (10–170 kDa, Thermo Fisher Scientific). Transfer to an Immobilon^®-FL PVDF membrane (Millpore (U.K) Ltd, Herfordshire, UK) was performed using a Novex^® semi-dry blotter (Thermo Fisher Scientific). VRC01 antibody was detected by using a 1:5000 dilution of an AP-conjugated Rabbit Anti-Human IgG H&L (Abcam). Bands were developed using the alkaline phosphate substrate BCIP/NBT kit (Thermo Fisher Scientific).

The Bio-Dot^® Apparatus (Bio-Rad) was used according to manufacturer’s instructions. 50 μL of sample supernatant from the deep-well plate expression was loaded into individual wells. A vacuum was applied until all the liquid had passed through the membrane. The VRC01 antibody was detected by using a 1:2000 dilution of an AP-conjugated Rabbit Anti-Human IgG H&L (Abcam, Cambridge, UK). Spots were developed using the alkaline phosphate substrate BCIP/NBT kit (Thermo Fisher Scientific).