MOESM1 of Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond

Additional file 1: Figure S1. Isoelectric focusing gel of sdAb. Measurement of isoelectric point using isoelectric focusing gel electrophoresis. Each well was loaded 10 µg of sample, except 4 µg of lane 5 sample was loaded. Lanes 1 and 10 represent the Serva pI marker (pH 3-10 from Life technologies Inc). Lane 2: Ac+neg: lane 3: AC+neg2; lane 4: AC+; lane 5: A3+; lane 6: A3+neg; lane 7: G2+; Lane 8: G2+neg; Lane 9: G2+neg2

MOESM3 of Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond

Additional file 3: Figure S3. Sequence alignment of sdAb AC and variants. Sequence alignment of the SEB binding sdAb AC, AC+, AC+neg and AC+neg2 using MultAlin [29]. The initial two amino acids (MA) and the amino acids added due to the restriction sites and the His-tag are not show above (AAALEHHHHHH)

MOESM4 of Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond

Additional file 4: Figure S4. Molecular weight and purity assessment of sdAb. Figure S4. Assessment of purity for purified single domain antibodies on gel electrophoresis. The virtual gel was obtained from Experion Pro260 chip (Bio-Rad laboratories). Approximately 200 µg/mL for each protein sample was used. The peak density of purified single domain antibodies as indicated by the blue arrow is >95 %. The rest of the bands represent high and low markers and internal systematic bands as indicated by the magenta arrows and described as such in the manufacturer’s protocol (Bio-Rad). Sample order is as follows, Lane L: Molecular marker Ladder. L1: ACneg; L2: AC+neg; L3: AC+neg2; L4: AC+;L5: A3+; L6: A3+neg; L7: G2+; L8: G2+neg; L9: G2+neg2; L10: G2

A New Family of Pyridine-Appended Multidentate Polymers As Hydrophilic Surface Ligands for Preparing Stable Biocompatible Quantum Dots

The growing utility of semiconductor quantum dots (QDs) in biochemical and cellular research necessitates, in turn, continuous development of surface functionalizing ligands to optimize their performance for ever more challenging and diverse biological applications. Here, we describe a new class of multifunctional polymeric ligands as a stable, compact and high affinity alternative to multidentate thiolated molecules. The polymeric ligands are designed with a poly­(acrylic acid) backbone where pyridines are used as anchoring groups that are not sensitive to degradation by air and light, along with short poly­(ethylene glycol) (PEG) pendant groups which are coincorporated for aqueous solubility, biocompatibility and colloidal stability. The percentages of each of the latter functional groups are controlled during initial synthesis along with incorporation of carboxyl groups which serve as chemical handles for subsequent covalent modification of the QD surface. A detailed physicochemical characterization indicates that the multiple pyridine groups are efficiently bound on the QD surface since they provide for relatively small overall hydrodynamic sizes along with good colloidal stability and strong fluorescence over a wide pH range, under high salt concentration and in extremely dilute conditions at room temperature under room light over extended timeframes. Covalent conjugation of dyes and metal-affinity coordination with functional enzymes to the QD surfaces were also demonstrated. Biocompatibility and long-term stability of the pyridine polymer coated QDs were then confirmed in a battery of relevant assays including cellular delivery by both microinjection and peptide facilitated uptake along with intracellular single QD tracking studies and cytotoxicity testing. Cumulatively, these results suggest this QD functionalization strategy is a viable alternative that provides some desirable properties of both compact, discrete ligands and large amphiphilic polymers