Anti penta his hrp conjugate

DNA sequences encoding miniSOG and miniSOG-Jα were synthesized by PCR overlap-extension and inserted into a bacterial expression vector (pQE-80L, Qiagen) in-frame with an N-terminal His-tag. For expression, transformed DH10B E. coli cells (Life Technologies) were grown in a shaking incubator (250 rpm) at 37°C in LB supplemented with 100 μg ml⁻¹ ampicillin. When a cell density of OD₆₀₀ ~0.4 was reached, cultures were either immediately induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG, at final concentration of 1 mM), or transferred to a 25°C shaking incubator (250 rpm) and induced 30 min later with the same concentration of IPTG. After 5 hours of expression, cells were pelleted by centrifugation, and soluble and insoluble proteins were fractionated using the B-PER Bacterial Protein Extraction Reagent (Pierce) supplemented with protease inhibitor cocktail (Complete, EDTA-free, Roche), both according to the manufacturer’s protocol. Protein fractions were analyzed by Coomassie staining of SDS-PAGE gels and immunoblotting with Anti-PentaHis HRP Conjugate (Qiagen). Blots were stripped with Restore Western Blot Stripping Buffer (Pierce) before being re-probed with Streptavidin-HRP (Pierce). In all cases, apparent molecular weights were approximated by comparison with the Precision Plus Protein Dual-Color Standard (Bio-Rad).