recombinant egfp protein  (OriGene)

Journal: bioRxiv

Article Title: Amplicon/Protein Bead Display enables quantitative in vitro biochemistry at scale

doi: 10.64898/2026.05.28.728566

Figure Lengend Snippet: a . Schematic of DNA templates encoding expression of a 25-variant FLAG library expressed as SNAP and eGFP fusion proteins on APBs. b . Schematic of experiments to detect binding between bead-displayed FLAG epitopes and M2 anti-FLAG antibodies using a Cy5-labeled secondary antibody. c . Representative brightfield and fluorescence images of APBs after incubation with M2 and Cy5-labeled secondary antibodies reporting on per-bead expression levels (GFP intensities), and antibody binding (Cy5 intensities). d . Box plots of per-bead median Cy5 pixel intensities (arbitrary units) for blank and GFP-positive beads. e . Schematic of single-bead sorting and sequencing workflow for validating genotype-phenotype linkages. FACS plot (far left) displays measured GFP (x-axis) and Cy5 (y-axis) intensities for the 384 GFP-positive beads sorted into a multi-well plate. f . Bar plots of expected (based on Poisson loading at λ = 0.1) and observed fractions of single-variant beads in the GFP-positive library. Error bars represent the Poisson-derived standard deviation. g . Measured Cy5 versus eGFP fluorescence intensities for all beads displaying three representative FLAG variants. Circles indicate single-variant beads; X’s indicate multi-variant beads. Red dashed lines indicate median Cy5/GFP ratio; gray shading shows interquartile range [0.25,0.75]. h . Median Cy5/GFP ratios calculated from all beads bearing a given variant versus single-variant beads only. Error bars denote interquartile range [0.25, 0.75]. i . Coefficient of variation (CV%) of median Cy5/GFP ratios across 1,000 bootstrap samples as a function of beads sampled per variant. j . Sequence logo showing relative position-normalized Cy5/GFP ratios for single amino acid substitutions (A, L, or E) at each position in the FLAG epitope. Red letters indicate wildtype residues; grey letters indicate substitutions.

Article Snippet: A fluorescence standard curve for estimating protein concentration on beads was generated using vortexed emulsion droplets containing known concentrations of recombinant eGFP protein (Origene).

Techniques: Expressing, Variant Assay, Binding Assay, Labeling, Fluorescence, Incubation, Sequencing, Derivative Assay, Standard Deviation, FLAG-tag

Journal: bioRxiv

Article Title: PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized non-ribosomal proteins

doi: 10.64898/2025.12.17.694911

Figure Lengend Snippet: a , Workflow for the expression and purification of PURE proteins by the PURE system and their functional evaluation. Each his-tagged protein in a given subset was expressed using PUREfrex by adding the corresponding DNA template to the reaction and incubating at 37°C. Protein expression was confirmed by BODIPY-lysine fluorescence on SDS–PAGE. Reactions corresponding to each subset were pooled and purified using Ni-charged magnetic beads, followed by buffer exchange using mini dialysis devices and concentrated by ultra-filtration. For the functional assay, the purified subset was added to the corresponding ΔPURE reaction (a PURE reaction lacking the corresponding proteins) containing an eGFP template as a reporter for protein synthesis. Full PURE is included as the positive control, ΔPURE + purification control as the negative control, ΔPURE + EE subset as the concentration adjusted control (containing E. coli synthesized proteins at the same concentration as the PUREfrex-expressed subsets as determined by Bradford assay), and the ΔPURE + PE subset (PUREfrex expressed proteins). The fluorescence signal was measured using a plate reader. b , The table summarizes the PUREfrex reaction volumes used for each protein and the final volume and concentration obtained after purification of each pooled subset. c , SDS–PAGE analysis of individual PUREfrex reactions expressing PURE proteins. The gel shows the BODIPY-lysine fluorescence of each synthesized PURE protein. d , Functional assay result of each subset. The plot shows eGFP fluorescence in each reaction (n=2 for all conditions). e , Yield and rate of eGFP synthesis calculated from panel d.

Article Snippet: Different concentrations of recombinant eGFP (OriGene, catalog no. TP790050) were prepared by dilution in PBS and added to a PUREfrex reaction lacking a DNA template, as the eGFP signal is amplified in PURE compared to PBS.

Techniques: Expressing, Purification, Functional Assay, Fluorescence, SDS Page, Magnetic Beads, Buffer Exchange, Filtration, Positive Control, Control, Negative Control, Concentration Assay, Synthesized, Bradford Assay

Journal: bioRxiv

Article Title: PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized non-ribosomal proteins

doi: 10.64898/2025.12.17.694911

Figure Lengend Snippet: a , Titration of the different protein subsets in PURE. Each subset was titrated in its corresponding 10-fold diluted ΔPURE reaction (containing a 10-fold lower concentration of non-ribosomal proteins), and the optimal mixing ratio was determined based on the effect of each subset’s volume on the reaction rate. Orange dots show synthesis rate data points, and blue crosses represent their average. b , Reconstitution of PURE by combining functional subsets. After producing the five PURE synthesized protein subsets, a new PURE reaction was reconstituted by combining all subsets. The mixing ratios are indicated on the arrows. Subsequently, 2.6 µL of the reconstituted PURE protein solution was added to a ΔPURE reaction (lacking all non-ribosomal proteins) containing an eGFP template for a functional test. c , Functional assay result of reconstituted PURE assembled from the five subsets. The plot shows eGFP fluorescence generated in each reaction ( n = 2 for all conditions). Full PURE is included as the positive control, ΔPURE supplemented with a purification control as the negative control, and the ad-justed control was prepared from E. coli expressed proteins at equivalent concentrations, combined at the same ratios, and added to the ΔPURE reaction. d , Yield and rate of eGFP synthesis calculated from panel c. e , Single-reaction regeneration of PURE proteins in PURE. All 36 non-ribosomal proteins were expressed together in a single PURE reaction by including all DNA templates encoding the respective proteins. For EF-Tu, a 35-fold excess of DNA template was used, and for EF-Ts, EF-G, and T7 RNAP, a 5-fold excess was used. After incubation at 37°C, magnetic beads were used for purification, followed by buffer exchange and concentration. Subsequently, 2.6 µL of the single-reaction regenerated PURE was added to a ΔPURE reaction (lacking all non-ribosomal proteins) containing an eGFP template for a functional test. f , SDS–PAGE of single-reaction regenerated PURE proteins visualized by BODIPY-lysine fluorescence. g , Coomassie-stained SDS-PAGE of final obtained single-reaction regenerated PURE proteins next to concentration adjusted E. coli expressed full PURE. h , Functional assay result of single-reaction regenerated PURE. The plot shows eGFP fluorescence generated in each reaction ( n = 2 for all conditions). Full PURE is included as the positive control, ΔPURE supplemented with a purification control as the negative control, and the adjusted control was prepared from homemade full PURE diluted to the same final total concentration and added to the ΔPURE reaction. i , Yield and rate of eGFP synthesis calculated from panel h.

Article Snippet: Different concentrations of recombinant eGFP (OriGene, catalog no. TP790050) were prepared by dilution in PBS and added to a PUREfrex reaction lacking a DNA template, as the eGFP signal is amplified in PURE compared to PBS.

Techniques: Titration, Concentration Assay, Functional Assay, Synthesized, Fluorescence, Generated, Positive Control, Purification, Control, Negative Control, Incubation, Magnetic Beads, Buffer Exchange, SDS Page, Staining

Journal: The Journal of Infectious Diseases

Article Title: Role of Flotillin-1 In Listeria monocytogenes Cell-to-Cell Spreading

doi: 10.1093/infdis/jiaf452

Figure Lengend Snippet: Localization of flotillin-1 during Listeria monocytogenes infections. A . Cells infected with L. monocytogenes for 8 hours were fixed and stained with rabbit polyclonal flotillin-1 targeting antibodies, Alexa Fluor 594 phalloidin to visualize F-actin, and 4',6-diamidino-2-phenylindole [DAPI] to stain for host and bacterial DNA. Boxed regions illustrate spreading events wherein flotillin was localized to invaginations at cell-to-cell spreading sites but not to protrusions extending into open space. B . Jeg3 cells transfected with green fluorescent protein (GFP)­–flotillin-1 were combined wild-type Jeg3 cells previously infected with L. monocytogenes. After 9 hours of total infection the cells were fixed and stained with Alexa Fluor 594 to visualize F-actin and DAPI to visualize host and bacterial DNA showing GFP–flotillin-1 localization specifically at the invaginations. A’, B’, Zoomed insets of boxed regions. C, Whole Jeg3 cell lysates from uninfected and infected cells were probed for endogenous flotillin-1. α-Tubulin is shown as a loading control. Scale bars represent 5 µm in the zoomed-out images and 2.25 µm in the zoomed insets; open arrowheads, L. monocytogenes protrusions; and closed arrowheads, invaginations.

Article Snippet: DNA constructs used in this study included green fluorescent protein (GFP)–flotillin-1 (Origene), tetracycline reponse element-enchanced green fluorescent protein (TRE-eGFP)-F1C and TRE-eGFP-F1A (gifts from Linda DeGraffenried; Addgene plasmids 211457 and 211458), and mCherry–caveolin-1 (gift from Michael Davidson; Addgene plasmid 55008).

Techniques: Infection, Staining, Transfection, Control

Journal: The Journal of Infectious Diseases

Article Title: Role of Flotillin-1 In Listeria monocytogenes Cell-to-Cell Spreading

doi: 10.1093/infdis/jiaf452

Figure Lengend Snippet: Cell-to-cell spreading assays using constructs in competition with endogenous flotillin-1 for palmitoylation. A, Control (F1A) or competitive inhibiting (F1C) peptides used in cell-to-cell spreading assays. CellTracker Blue–stained cells that were initially infected with Listeria monocytogenes (sending cells) were combined with uninfected Jeg3 cells transfected with tetracycline reponse element-enchanced green fluorescent protein (TRE-eGFP)-F1A/F1C constructs (receiving cells). Following 6-hour incubations, the cells were fixed and stained with Alexa Fluor 594 phalloidin. Arrowheads point to actin-rich structures counted as a measure of cell-to-cell spreading. Scale bar represents 5 µm. A’, Zoomed insets of boxed regions for the control peptide ( top ) and the palmitoylation competitive peptide ( bottom ) highlight differences in the abundance of actin-rich structures. The red channel was enhanced to ease the visualization of actin. Scale bar represents 2.25 µm. B, Actin-rich structures were counted in ≥50 receiving cells of the cell-to-cell spreading assays in 3 separate experiments. Competitive inhibiting peptide counts were normalized to the wild type. A 2-tailed t test with Welch correction was used to analyze the data, resulting in significantly fewer actin-rich structures in the F1C than in the F1A assays. *** P < .001.

Article Snippet: DNA constructs used in this study included green fluorescent protein (GFP)–flotillin-1 (Origene), tetracycline reponse element-enchanced green fluorescent protein (TRE-eGFP)-F1C and TRE-eGFP-F1A (gifts from Linda DeGraffenried; Addgene plasmids 211457 and 211458), and mCherry–caveolin-1 (gift from Michael Davidson; Addgene plasmid 55008).

Techniques: Construct, Control, Staining, Infection, Transfection

Journal: The Journal of Infectious Diseases

Article Title: Role of Flotillin-1 In Listeria monocytogenes Cell-to-Cell Spreading

doi: 10.1093/infdis/jiaf452

Figure Lengend Snippet: The effects of small interfering RNA (siRNA) flotillin-1 knockdown (KD) in Jeg3 cells on the spreading events of Listeria monocytogenes. A, Control Jeg3 cells exposed to scrambled nontargeting siRNA (control) or siRNA flotillin-1 were subjected to cell-to-cell spreading assays, as described elsewhere. Following 6-hour incubations, the cells were fixed and stained with Alexa Fluor 594 phalloidin. The actin-rich structures were enumerated as a measure of cell-to-cell spreading (some of the actin-rich structures are indicated by arrowheads). Scale bar represents 5 µm. Abbreviation: GFP, green fluorescent protein. A’, Zoomed insets of boxed regions for control ( top ) and siRNA flotillin-1 ( bottom ) illustrating differences in the abundance of actin-rich structures. Scale bar represents 5 µm. B, Actin-rich structures were counted in ≥50 receiving cells of the mixed cell assays in 3 independent experiments with the siRNA counts normalized to the scramble controls. A 2-tailed t test with Welch correction was used to analyze the data, which resulted in significantly fewer actin-rich structures in the siRNA assays compared with control siRNA. *** P < .001. C, Western blot showing the efficacy of the flotillin-1 KD compared with controls. α-Tubulin was used as a loading control. Endogenous flotillin-2 expression levels were also probed for during flotillin-1 KD.

Article Snippet: DNA constructs used in this study included green fluorescent protein (GFP)–flotillin-1 (Origene), tetracycline reponse element-enchanced green fluorescent protein (TRE-eGFP)-F1C and TRE-eGFP-F1A (gifts from Linda DeGraffenried; Addgene plasmids 211457 and 211458), and mCherry–caveolin-1 (gift from Michael Davidson; Addgene plasmid 55008).

Techniques: Small Interfering RNA, Knockdown, Control, Staining, Western Blot, Expressing

Journal: The Journal of Infectious Diseases

Article Title: Role of Flotillin-1 In Listeria monocytogenes Cell-to-Cell Spreading

doi: 10.1093/infdis/jiaf452

Figure Lengend Snippet: Flotillin-1 further hinders Listeria monocytogenes cell-to-cell spreading in cells devoid of caveolin-1 (Cav-1). A, Colocalization of green fluorescent protein (GFP)–flotillin-1 and mCherry–Cav-1 at invaginations were analyzed in cells infected with L. monocytogenes . Cells were also stained with Alexa Fluor 405 phalloidin to label F-actin. Arrowheads indicate invagination sites. Scale bars represent 5 µm in the zoomed-out images and 2.25 µm in the zoomed insets. B, Cell-to-cell spreading events between the 3 experimental conditions where wild-type (WT) CellTracker Blue­–stained (CTB) HeLa cells previously infected with GFP– L. monocytogenes were combined with HeLa, HeLa Cav-1 knockdown (KD) or HeLa Cav-1 KD small interfering RNA (siRNA) flotillin-1 cells for 6 hours. Cells were fixed, and F-actin was stained with Alexa Fluor 594 phalloidin. Scale bar represents 20 µm. C, Images of the infection foci measured among the 3 experimental conditions. GFP– L. monocytogenes was thresholded ( left ) and the infection foci masked ( right ) using Metamorph software to visualize the cell-to-cell spreading events. D, Quantification of the infection foci of HeLa, HeLa Cav-1 KD and HeLa Cav-1 KD siRNA flotillin-1 cells. At least 40 cells were measured in each experimental condition from 3 independent replicates. The HeLa Cav-1 KD (30.5% of the WT area) and HeLa Cav-1 KD siRNA flotillin-1 (16% of the WT area) values were normalized to WT HeLa spreading events and compared using 1-way analysis of variance (ANOVA) with Tukey test. *** P < .001. Scale bar represents 20 µm. E, Enumeration of cells in which bacteria were restricted to the initially infected HeLa cell to illustrate unsuccessful spreading. At least 40 cells were observed from 3 independent replicates in each experimental condition and compared using 1-way ANOVA with Tukey test. WT HeLa cells had 0% blocked cell-to-cell spreading, while an average of 20% of the HeLa Cav-1 KD cells and 40% of the HeLa Cav-1 KD siRNA flotillin-1 cells had blocked spreading events. * P < .05; ** P < .01; *** P < .001. F, Whole-cell lysates from HeLa Cav-1 KD/flotillin-1 KD and HeLa Cav-1 KD (scrambled nontargeting siRNA), and HeLa cells were collected and probed for endogenous flotillin-1 using rabbit polyclonal antibodies. α-Tubulin was used as a loading control.

Article Snippet: DNA constructs used in this study included green fluorescent protein (GFP)–flotillin-1 (Origene), tetracycline reponse element-enchanced green fluorescent protein (TRE-eGFP)-F1C and TRE-eGFP-F1A (gifts from Linda DeGraffenried; Addgene plasmids 211457 and 211458), and mCherry–caveolin-1 (gift from Michael Davidson; Addgene plasmid 55008).

Techniques: Infection, Staining, Knockdown, Small Interfering RNA, Software, Bacteria, Control