Anti 6his

Cells were grown to midlog phase, pelleted, washed with dH₂O, and frozen in liquid nitrogen. Pellets were resuspended in 350 μl IPH50 [50 mM Tris pH 8.0, 50 mM NaCl, 5 mM EDTA, 0.5% NP-40, 5 mM β-mercaptoethanol, protease inhibitor cocktail (Sigma)]. Cells were lysed by adding glass beads (Sigma) to the resuspended pellets followed by four working cycles of 1 min in a bullet blender (Next Advance). The lysates were cleared by two centrifugations of 5 min and 15 min at 1000 × g and 14,000 × g, respectively, at 4°. Immunoprecipitations were performed at 4° adding the appropriate antibodies for 2 h. The antibodies were collected on protein A/G agarose (Santa Cruz) or on magnetic beads (BIO-RAD) for 1 hr, and washed three times with IPH50 and resuspended in 35 μl Laemmli buffer. Standard procedures for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting were followed to transfer proteins from gels to a polyscreen PVDF membrane (Millipore). Membranes were blotted with the primary antibodies. Antibodies were detected using SuperSignal West Pico (Thermo Scientific) and LAS 4000 (GE Healthcare). Antibodies used in this study were: anti-HA (12CA5, Roche), anti-MYC (9E10, Roche), anti-V5 (Invitrogen/Millipore), anti-3Flag (Sigma), and anti-6His (Sigma).

SDS–PAGE was performed on Bio-Rad Mini-PROTEAN systems using standard protocols. For immunostaining, proteins were transferred onto 0.2-μm nitrocellulose membranes (Amersham Protran) Immunoblots were probed with primary antibodies and goat secondary antibodies coupled to alkaline phosphatase and developed in alkaline buffer in presence of 5-bromo-4-chloro-3- indolylphosphate and nitro-blue tetrazolium. The anti-HA (HA-7 clone, Sigma Aldrich), anti-Flag (Sigma Aldrich, A2220-1ml or F3165-.2MG), anti-StrepII (Bio-Rad), anti VSV-G (Sigma Aldrich, SAB4200695-100UL), anti-6His (Sigma Aldrich, SAB1305538-400UL), anti-StrepII (Biorad MCA2489), anti-GFP (Thermofischer, MA5-15256), RNA-pol II (Ozyme 664906) monoclonal antibodies, rabbit polyclonal mcherry antibodies (OriGene TA150125) mouse secondary antibodies (Interchim 115-055-003) and rat secondary antibodies (Interchim115-055-045) were purchased as indicated. Synthetics polyclonalrabbit antibodies were designed by GeneScript for Hcp (epitope: SVGGHTAERVEHSDC) and TssM detection (epitope: CAPAQAAAPAKTENP).

Protein quantification of the chromatographic fractions was performed using bicinchoninic acid protein micro-assay (BCA kit, Sigma). Twenty-five microliters samples were incubated with 200 μL of BCA working reagent and plate was incubated at 37°C for 30 min. The absorbance was measured at 562 nm.
Fractions obtained from chromatographic experiments were analyzed by SDS-PAGE under reducing conditions over 12% polyacrylamide gels. Supernatant and flow through fractions were 5x concentrated by TCA precipitation. Precision plus protein prestained standards (BIO-RAD) were used as molecular weight ladder.
To analyse the efficiency of the yeast cells P. pastoris to produce the scFv fragments into the broth medium, 50 μL samples from day 1 to day 5 were blotted on a nitrocellulose membrane using Bio-Dot Microfiltration Instrument (BIO-RAD). The membrane was blocked with a blocking buffer (TBS Tween with 3% milk powder) for 2 hours. The membrane was again washed twice with TBS-tween, and then incubated with primary antibody (Anti 6His, SIGMA) at 1:1500 dilution overnight at 4°C. Membranes were then washed and probed with a secondary antibody (anti-mouse IgG-HRP Cell Signaling Technology) at 1:5000 dilution.
Colorimetric analysis was performed using Opti4CN (BIO-RAD) kit by gently shaking until color develops.