Ligation of solution phase DNA to a bead-immobilized vector.

<p>(<b>A</b>) A fluorescence-based assay for determining extent of ligation. Beads with immobilized vector are incubated with a Alexa 488 fluorescent oligo. The extent of ligation is measured via flow cytometry. Bead loading was at 1 ng vector DNA (pHISZ)/ug bead vector. Positive: Beads in which pHISZ vector is fully fluorescently labeled. Ligation: Beads after ligation. Negative: Beads in which pHISZ vector is not fluorescently labeled. The extent of ligation f is measured as a percentage of the Positive signal. (<b>B</b>) Extent of ligation is reduced at high bead loadings.</p

Bead-bead subcloning of cancer genes into multiple expression vectors.

<p>The ectodomains of three cancer antigens (TNF, CA9 ectodomain, and PSMA ectodomain) were subcloned via bead-bead subcloning from an <i>E. coli</i> expression vector into expression vectors for <i>S. carnosus, P. pastoris</i>, and CHO. (A) PCR screens of pLentiHAp transformants show correct sizes for 10/10. (B) Expression of target proteins on the surface of <i>S. carnosus</i> using the surface display vector pSCEM2. The surface expression is quantified via flow cytometry as HSA-Alexa647 binding to ABP, which is co-expressed with the target protein. (C) Expression of target proteins from the CHO expression vector pLenitHAp. Western blotting (anti His6) of CHO cell lysates. (D) Expression of target proteins from the <i>P. pastoris</i> expression vector pPICZap. Western blotting (anti His6) of <i>P. pastoris</i> cell lysates.</p

The presence of magnet increases the extent of ligation in bead-bead subcloning.

<p>Flow cytometry of fluorescent-labeled beads allows quantification of bead-bead ligations. Insert beads are red-labeled (Alexa488 label) and acceptor-vector beads are green-labeled (Alexa647 label). Successful bead-bead ligations appear at high FL-1 and FL-6 readouts (boxed). The insert is ITGA2b (1 350 bp) and the acceptor vector is pLenti1 (8 180 bp). (A) Ligation in the absence of magnet. Approximately 0.5% of acceptor beads are ligated. (B) Ligation in the presence of magnet. Approximately 7% of the acceptor beads are ligated, a 14-fold increase in extent of ligation.</p

Statistics of automated bead-bead subcloning of 95 target genes.

<p>Statistics of automated bead-bead subcloning of 95 target genes.</p

Ligation efficiencies of bead-based subcloning strategies.^*

*<p> <i>Vector and donor beads were loaded at 0.5 ng DNA/µg bead.</i></p

Structure analysis of epitopes.

<p>3D structures of protein targets with highlighted linear epitopes in green where the corresponding antibody fraction showed bands of correct molecular weight in Western blot analysis and in red where the antibody fraction did not bind the target protein. SYNJ2BP (2eno.pdb), TYMP (2jof.pdb), WARS (1r6t.pdb), CD4 (1wio.pdb), OTC (1oth.pdb), CRABP2 (2g7b.pdb), PDXP (2cft.pdb) and EGFR (3njp.pdb).</p

Classification of all protein-coding genes based on their expression in human urinary bladder tissue.

<p>(A) Scatterplot showing the FPKM values of all 20,344 protein-coding genes in the two urinary bladder samples, with each gene colored according to category. (B) Pie chart showing the distribution of all protein-coding genes into five categories based on transcript abundance and number of detected tissues, including expression in all 32 tissues (blue), mixed expression with genes expressed in a varying number of tissue (green), genes with elevated expression in urinary bladder (pink), not detected in urinary bladder (light grey), and not detected in any tissue (dark grey). The genes with elevated expression in urinary bladder are further subdivided depending on the degree of specificity as tissue enriched genes, group enriched genes and tissue enhanced genes in urinary bladder (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145301#pone.0145301.t001" target="_blank">Table 1</a>). (C) Pie chart showing the distribution of the fraction of expressed mRNA molecules, i.e. the sum of all FPKM values for each of the categories for the genes expressed in urinary bladder, using the same color codes. (D) Network plot of the urinary bladder enriched gene (red) and group enriched genes (orange). Orange circle nodes represent a shared group of expressed genes and are connected to the respective enriched tissues (grey circles). The size of each orange node is related to the square root of the number of genes enriched in a particular combination of tissues.</p

Immunohistochemistry-based protein profiling of elevated genes in urinary bladder that are expressed in umbrella cells.

<p>Of the 90 elevated genes in urinary bladder, UPK1A (group enriched gene), UPK2 (tissue enriched gene), UPK3A (group enriched gene) and UPK3B (group enriched gene) are localized specifically to umbrella cells in urothelium. All images are from the Human Protein Atlas and the title of each images show the respective gene and antibody names.</p

Immunohistochemistry-based protein profiling of elevated genes in urinary bladder that are expressed in intermediate/basal layer cells.

<p>Of the 90 elevated genes in urinary bladder, KRT17 (tissue enhanced gene), PCP4L1 (tissue enhanced gene) and ATP1A4 (tissue enhanced gene) are localize specificity in intermediate/basal layer cells in urothelium. All images are from the Human Protein Atlas. Titles on each images are gene and antibody names of concerned proteins.</p

Fractionation of polyclonal antibodies.

<p>(A) Illustration depicting the scheme for decomposition of polyclonal antibodies into fractions targeting conformational and linear epitopes. First columns containing the antigen’s protein tag, peptides corresponding to previously mapped linear epitopes, a mix of overlapping peptides covering the antigen sequence and the antigen used for immunization are serially connected. Polyclonal serum is run through the columns where anti-tag antibodies are depleted by the first column, the peptide columns capture antibodies targeting the different linear epitopes and the antigen column binds the remaining antibodies that are targeting conformational epitopes. The columns are then separated from each other and the different antibody fractions are eluted in parallel. (B) An example of results from validation of a polyclonal antibody and antibody fractions towards the target protein tryptophanyl-tRNA synthetase. The left column shows epitope mapping confirming the peptide specificity of the different fractions. The middle column shows the relative antibody amount in each fraction. The right column shows the ability of each antibody fraction to detect a band of expected molecular weight, indicated by an arrow, in Western blots assays with RT-4 and U-251 MG lysates.</p