affigel 10/15 resin  (Bio-Rad)

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Schematic of gene expression output of the circadian clock under Constant Light conditions. Under Constant Light intensity (dashed navy blue line), dawn gene expression (dashed maroon line) and dusk gene expression (solid green line) display oscillatory patterns, peaking at subjective dawn and subjective dusk, respectively. The Kai post-translational oscillator generates oscillations in the levels of phosphorylated RpaA (RpaA ∼ P) and the binding of RpaA to DNA (black dotted line), with the peak amplitude at subjective dusk. ( B ) Solar irradiance measurements in units of watts m − 2 at 342.5 meters above sea level in Madison, WI, on a clear day (3/23/13, dark blue), and a day on which fluctuations in cloud cover generated rapid changes in light intensity (4/12/14, light blue) . Examples of a ‘High Light pulse,’ ‘Shade pulse,’ and ‘Sunset’ are indicated. ( C ) Schematic of the regulation of circadian gene expression. RpaA phosphorylation state converts timing information from the Kai oscillator to changes in gene expression by directly binding and activating a subset of dusk genes, indirectly activating the remainder of the dusk genes, and indirectly repressing the dawn genes. High Light Pulse conditions cause dephosphorylation of RpaB , but the effects of conditions like Sunset or Shade on RpaB are unknown. It is unclear whether natural fluctuations in light directly affect the clock and its output pathways and how light-induced changes in RpaB activity might be involved.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Expressing, Binding Assay, Generated, De-Phosphorylation Assay, Activity Assay

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Phosphorylation dynamics of RpaA under Low Light vs High Light pulse. Relative levels of phosphorylated RpaA were measured using Phos-tag Western blotting (left y-axis) in cells grown under Low Light conditions (black squares, see for Setup) or High Light pulse conditions (orange triangles, see for Setup). Each point represents the average of values measured in two independent Western blots, with error bars displaying the range of the measured values. See Materials and methods, Measurement of RpaA ∼ P and RpaB ∼ P levels for more details. Data is available in . The light profile for each condition is plotted as dashed lines of the same color with values corresponding to the right y-axis. ( B ) Phosphorylation dynamics of RpaA under Clear Day (magenta triangles, see for Setup) vs Shade pulse (gray circles, see for Setup) conditions, measured and plotted as in ( A ). ( C ) The change in enrichment of RpaA at a given peak upstream of a dusk gene (x-axis) and the corresponding change in expression of the downstream dusk gene (y-axis) from the original condition after 60 min in High Light (orange triangles) or Shade (gray circles), plotted for the 56 dusk genes with detectable RpaA peaks in their promoters. The correlation coefficient for the data taken in High Light and Shade conditions is indicated above the plot. See Materials and methods, ChIP-seq analysis for more details. Data is available in . ( D ) The change in enrichment of RpaA at a given peak upstream of a dusk gene (x-axis) and the corresponding change in RNAP enrichment upstream of the same gene (y-axis) from the original condition after 60 min in High Light (orange triangles) or Shade (gray circles), plotted for the 33 dusk genes with detectable RpaA and RNAP peaks in their promoters. The correlation coefficient for High Light and Shade data is indicated above the plot. See Materials and methods, ChIP-seq analysis for more details. ( E ) Model of regulation of dusk genes by RpaA under naturally-relevant conditions. The Kai PTO controls levels of RpaA ∼ P independent of changes in environmental light intensity. Changes in light intensity regulate the recruitment of RpaA ∼ P with RNAP to dusk genes to control their expression in response to environmental perturbations. 10.7554/eLife.32032.020 Figure 4—source data 1. Quantification of relative RpaA∼P levels. 10.7554/eLife.32032.021 Figure 4—source data 2. List of RpaA peaks, gene targets, and quantification of enrichment under High Light pulse and Shade pulse conditions.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Western Blot, Expressing, ChIP-sequencing

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A )-( C ) Normalized ChIP-seq signal of RpaA (red), RpaB (blue), RNAP (green) and mock IP (black) upstream of the ( A ) the representative dusk gene Synpcc7942_2267 , ( B ) the kaiBC operon and ( C ) another representative dusk gene, digC , at 8 hr since dawn in Low Light. The chromosomal position of the gene is located on the plot with a gray bar with an arrow indicating directionality of the gene. The location of RpaA and RNAP peaks are indicated on top of the plot with red (RpaA) and green (RNAP) bars. No RpaB peaks were found upstream of these genes. No RNAP peak was found upstream of kaiB or digC . See Materials and methods, ChIP-seq analysis for more details. ( D )-( F ) Changes in enrichment of RpaA (red) and RNAP (green) and downstream gene expression (black) after exposure to the High Light pulse (triangles) or the Shade pulse (circles) for ( D ) Synpcc7942_2267 , ( E ) the kaiBC operon, and ( F ) digC . See Materials and methods, ChIP-seq analysis for more details. ( G )-( L ) Gene expression dynamics of Synpcc7942_2267 ( G,J ) kaiB ( H,K ) and digC ( I,L ) under Low Light vs High Light pulse ( G–I ) and Clear Day vs Shade pulse ( J–L ) conditions. RpaA binding does not change upstream of the dusk genes Synpcc7942_2267 and kaiB after changes in light ( D,E ), and the expression of these genes does not change significantly in response to changes in light intensity (G,J;H,K). In contrast, RpaA binding changes significantly ( F ) upstream of the light-responsive dusk gene digC ( I,L ).

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: ChIP-sequencing, Expressing, Binding Assay

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Phosphorylation dynamics of RpaB under Low Light vs High Light pulse. Relative levels of phosphorylated RpaB were measured using Phos-tag Western blotting (left y-axis) in cells grown under Low Light conditions (black squares, see for Setup) or High Light pulse conditions (orange triangles, see for Setup). Each point represents the average of values measured in two independent Western blots, with error bars displaying the range of the measured values. See Materials and methods, Measurement of RpaA ∼ P and RpaB ∼ P levels for more details. Data is available in . The light profile for each condition is plotted as dashed lines of the same color with values corresponding to the right y-axis. ( B ) Phosphorylation dynamics of RpaB under Clear Day (magenta triangles, see for Setup) vs Shade pulse (gray circles, see for Setup) conditions, measured and plotted as in ( A ). ( C ) The change in enrichment of RpaB at a given peak upstream of a dusk gene (x-axis) and the corresponding change expression of the downstream dusk gene (y-axis) from the original condition after 60 min in High Light (orange triangles) or Shade (gray circles), plotted for the 42 dusk genes with detectable RpaB peaks in their promoters. The correlation coefficient for High Light and Shade data is indicated above the plot. See Materials and methods, ChIP-seq analysis for more details. Data is available in . ( D ) The change in enrichment of an RpaB at a given peak upstream of a dusk gene (x-axis) and the corresponding change in RNAP enrichment upstream of the same gene (y-axis) from the original condition after 60 min in High Light (orange triangles) or Shade (gray circles), plotted for the 27 dusk genes with detectable RpaB and RNAP peaks in their promoters. he correlation coefficient for High Light and Shade data is indicated above the plot. See Materials and methods, ChIP-seq analysis for more details. ( E ) Model of regulation of dusk genes by RpaB under naturally-relevant conditions. Changes in light regulate RpaB ∼ P levels. RpaB ∼ P binds with RNAP to dusk genes to control their expression in response to environmental perturbations. 10.7554/eLife.32032.026 Figure 5—source data 1. Quantification of relative RpaB∼P levels. 10.7554/eLife.32032.027 Figure 5—source data 2. List of RpaB peaks, gene targets, and quantification of enrichment under High Light pulse and Shade pulse conditions.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Western Blot, Expressing, ChIP-sequencing

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Changes in enrichment of RpaB upstream of dusk genes during High Light pulse conditions (left heat map) and corresponding changes in RNAP enrichment upstream of the same gene (right heat map) for the 27 dusk genes with RpaB and RNAP peaks in their promoters. ChIP enrichment is expressed as the log 2 fold change of enrichment in High Light from enrichment at 8 hr since dawn in Low Light conditions (time zero). RpaB and RNAP peaks upstream of the same dusk gene are aligned horizontally in the two heat maps. See Materials and methods, ChIP-seq analysis for more details. ( B ) Changes in enrichment of RpaB upstream of dusk genes during Shade pulse conditions (left heat map) and corresponding changes in RNAP enrichment upstream of the same gene (right heat map). ChIP enrichment is expressed as the log 2 fold change of enrichment in Shade from enrichment at 8 hr since dawn in Clear Day conditions (time zero). RpaB RNAP peaks upstream of the same dusk gene are aligned horizontally in the two heat maps. Peaks have the same order in ( A ) and ( B ). See Materials and methods, ChIP-seq analysis for more details. The correlation between RpaA and RNAP enrichment change upstream of dusk gene expressions reported in also holds after 15 min of exposure to High Light or Shade (High Light correlation = 0.22, Shade correlation = 0.42).

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: ChIP-sequencing

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Number of dusk gene targets of RpaA only (red), RpaB only (blue), RpaA and RpaB (yellow), or neither (black). Target genes of binding sites of RpaA and RpaB were determined using chromatin immunoprecipitation followed by sequencing under several different light conditions (see Materials and methods, ChIP-seq analysis, for more details. See or for full lists of RpaA and RpaB peaks associated with dusk genes). ( B ) Light-responsive changes in gene expression of dusk genes. For each dusk gene, we calculated the maximal log 2 change in expression during the High Light pulse (x-axis) or Shade pulse (y-axis) from 8 hr since dawn in the Low light or Clear day conditions, respectively, using the data from . ( C ) Normalized ChIP-seq signal of RpaA (red), RpaB (blue), RNAP (green) and mock IP (black) upstream of the dusk sigma factor gene rpoD6 at 8 hr since dawn in Low Light. The chromosomal location of the gene is located on the plot with a gray bar with an arrow indicating directionality of the gene. The location of RpaA, RpaB, and RNAP peaks are indicated on top of the plot with red (RpaA), blue (RpaB), and green (RNAP) bars. See Materials and methods, ChIP-seq analysis for more details. ( D ) Changes in enrichment upstream of rpoD6 of RpaA (red), RpaB (blue), and RNAP (green) and changes in rpoD6 gene expression (black) after exposure to the High Light pulse (triangles) or the Shade pulse (circles). See Materials and methods, ChIP-seq analysis for more details.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Sequencing, ChIP-sequencing, Expressing

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A )-( C ) Normalized ChIP-seq signal of RpaA (red), RpaB (blue), RNAP (green) and mock IP (black) upstream of the sigma factor genes ( A ) rpoD6 , ( B ) rpoD5 , and ( C ) sigF2 . The location of the gene is located on the plot with a gray bar with an arrow indicating directionality of the gene. The location of RpaA, RpaB, and RNAP peaks are indicated on top of the plot with red (RpaA), blue (RpaB), and green (RNAP) bars. See Materials and methods, ChIP-seq analysis for more details. ( D )-( F ) Changes in enrichment of RpaA (red), RpaB (blue), and RNAP (green) and downstream sigma factor gene expression (black) after exposure to the High Light pulse (triangles) or the Shade pulse (circles) upstream of rpoD6 ( D ), rpoD5 ( E ), and sigF2 ( F ). See Materials and methods, ChIP-seq analysis for more details. ( G )-( L ) Gene expression dynamics of rpoD6 ( G,J ), rpoD5 ( H,K ), and sigF2 ( I,L ) under Low Light vs High Light pulse ( G )-( I ) and Clear Day vs Shade pulse ( J )-( L ) conditions. RpaA and RpaB binding changes in a correlated manner upstream of these genes. RpaA and RpaB binding also correlates with changes in RNAP enrichment and sigma factor expression levels.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: ChIP-sequencing, Expressing, Binding Assay

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Average expression profiles of genes belonging to the Early dusk gene cluster under Clear Day (magenta) and Shade pulse (gray) conditions (left y-axis). Dusk genes were grouped using k-means clustering of their normalized expression dynamics in response to the four light conditions of this study and perturbations of RpaA activity in Constant Light conditions ( , ), and clusters were named based on their order of activation. See Materials and methods - K-means clustering for more details, and for full lists of genes in each cluster. The number of genes within the cluster, as well as the number of genes with an RpaA or RpaB peak in their promoters (targets) is listed. The expression values of each gene across all four light conditions in this work were normalized to a range of 0 to 1, and the normalized expression values were averaged within each cluster (solid lines). The shaded region on the plot indicates the standard deviation of the normalized expression values within the cluster. The light intensity profile for each condition is plotted as dashed lines in the same color with values corresponding to the right y-axis. ( B ) Average expression profiles of genes belonging to the Middle dusk gene cluster under Clear Day (magenta) and Shade pulse (gray) conditions (left y-axis), presented as in ( A ). ( C ) Average expression profiles of genes belonging to the Late dusk gene cluster under Clear Day (magenta) and Shade pulse (gray) conditions (left y-axis), presented as in ( A ). 10.7554/eLife.32032.032 Figure 7—source data 1. Lists of genes belonging to the Early, Middle, and Late dusk clusters, and scaled gene expression values.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Expressing, Activity Assay, Activation Assay, Standard Deviation

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Normalized RpaA ∼ P levels (left plot) and RpaB ∼ P levels (right plot) under Clear Day (magenta) and Shade pulse (gray) conditions used as input for mathematical models of dusk gene expression. RpaA ∼ P or RpaB ∼ P levels from all four light conditions were normalized to a range of 0 to 1. ( B ) In the ‘RpaA and RpaB’ models, RpaA ∼ P and RpaB ∼ P jointly activate the expression of the Early (E), Middle (M), or Late (L) cluster. See Materials and methods - Mathematical modeling for more details. ( C ) Simulations of the best fit ‘RpaA and RpaB’ model for the Early dusk genes. Average cluster expression data is shown as faded solid lines, and the best fit simulations are shown as dotted lines. Data for Clear Day conditions are plotted in magenta, and Shade pulse in gray. See Materials and methods - Mathematical modeling for more details. ( D ) Simulations of the best fit ‘RpaA and RpaB’ model for the Middle dusk genes, plotted as in ( C ). ( E ) Simulations of the best fit ‘RpaA and RpaB’ model for the Late dusk genes, plotted as in ( C ). ( F ) In the ‘Feedback’ models, another cluster activates or represses the expression of the Early (E), Middle (M), or Late (L) cluster alongside joint activation by RpaA ∼ P and RpaB ∼ P. ( G ) Simulations of the best fit ‘Feedback’ model for the Early dusk genes, plotted as in ( C ). In this model, Late cluster expression represses Early cluster expression alongside activation by RpaA ∼ P and RpaB ∼ P. ( H ) Simulations of the best fit ‘Feedback’ model for the Middle dusk genes, plotted as in ( C ). In this model, Late cluster expression activates Middle cluster expression alongside activation by RpaA ∼ P and RpaB ∼ P. ( I ) Simulations of the best fit ‘Feedback’ model for the Late dusk genes, plotted as in ( C ). In this model, Middle cluster expression activates Late cluster expression alongside activation by RpaA ∼ P and RpaB ∼ P.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Expressing, Activation Assay

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Normalized RpaA ∼ P levels under Clear Day (magenta) and Shade pulse (gray) conditions used as input for mathematical models of dusk gene expression. RpaA ∼ P levels from all four light conditions were normalized to a range of 0 to 1. ( B ) In the ‘RpaA-only’ models, RpaA ∼ P activates the expression of the Early (E), Middle (M), or Late (L) cluster. (C) Simulations (dotted lines) of best fit RpaA-only models for Clear Day and Shade pulse data (solid lines) for the Early (Left plot), Middle (middle plot), and Late (right plot) dusk genes. Data for Clear Day conditions are plotted in magenta, and Shade pulse in gray. ( D ) Normalized RpaB ∼ P levels under Clear Day (magenta) and Shade pulse (gray) conditions used as model input. ( E ) Model schematic. Dusk gene expression under Clear Day and Shade pulse conditions was modeled as an activation Hill function of RpaB ∼ P levels only. ( F ) Simulations (dotted lines) of best fit RpaB-only models for Clear Day and Shade pulse data (solid lines) for the Early (Left plot), Middle (middle plot), and Late (right plot) dusk genes. Data for Clear Day conditions are plotted in magenta, and Shade pulse in gray.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Expressing, Activation Assay

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: Fitting results. The definitions of the variables are given in Equations 1-3 , p. 1–3. The error is defined as the square root of the sum of the squared deviations between simulation and data.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques:

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Feedback model in which the expression of the Early dusk cluster is an activation Hill function of Middle gene expression and an activation Hill function of both RpaA ∼ P and RpaB ∼ P. The left plot shows normalized Middle cluster expression levels under Clear Day (magenta) and Shade pulse (gray) conditions used as model input in addition to the RpaA ∼ P and RpaB ∼ P dynamics shown in of the main text. The right plot shows the average expression values of the Early cluster data (solid transparent lines), and the simulation produced the best fit model (dotted lines). Data for Clear Day conditions are plotted in magenta, and Shade pulse in gray. ( B ) Feedback model in which Early gene cluster expression is a repression Hill function of Middle cluster expression levels and an activation Hill function of both RpaA ∼ P and RpaB ∼ P, presented as in ( A ). ( C ) Feedback model in which Early cluster expression is an activation Hill function of Late cluster expression levels and an activation Hill function of both RpaA ∼ P and RpaB ∼ P. The left plot shows normalized Late cluster expression levels under Clear Day (magenta) and Shade pulse (gray) conditions used as model input. The right plot shows the average expression values of the Early cluster data (solid transparent lines), and the simulation produced the best fit model (dotted lines). Data for Clear Day conditions are plotted in magenta, and Shade pulse in gray. ( D ) Feedback model in which Early cluster expression is a repression Hill function of Late cluster expression levels and an activation Hill function of both RpaA ∼ P and RpaB ∼ P, presented as in ( C ). A model with an incoherent feedforward architecture in which the Late cluster represses Early cluster expression ( D ) best recapitulates the difference of Early cluster responses to Shade and Clear Day Sunset. During the Shade pulse, the Late cluster levels do not reach high enough levels to inhibit Early cluster expression, but at Sunset in Clear Day, Late cluster levels reach high enough levels to repress the expression of the Early cluster.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Expressing, Activation Assay, Produced

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet: ( A ) Feedback model in which the expression of the Late dusk cluster is an activation Hill function of Early gene expression and an activation Hill function of both RpaA ∼ P and RpaB ∼ P. The left plot shows normalized Early cluster expression levels under Clear Day (magenta) and Shade pulse (gray) conditions used as model input in addition to the RpaA ∼ P and RpaB ∼ P dynamics shown in of the main text. The right plot shows the average expression values of the Middle cluster data (solid transparent lines), and the simulation produced the best fit model (dotted lines). Data for Clear Day conditions are plotted in magenta, and Shade pulse in gray. ( B ) Feedback model in which Late gene cluster expression is a repression Hill function of Early cluster expression levels and an activation Hill function of both RpaA ∼ P and RpaB ∼ P, presented as in ( A ). ( C ) Feedback model in which Late cluster expression is an activation Hill function of Middle cluster expression levels and an activation Hill function of both RpaA ∼ P and RpaB ∼ P. The left plot shows normalized Middle cluster expression levels under Clear Day (magenta) and Shade pulse (gray) conditions used as model input. The right plot shows the average expression values of the Late cluster data (solid transparent lines), and the simulation produced the best fit model (dotted lines). Data for Clear Day conditions are plotted in magenta, and Shade pulse in gray. ( D ) Feedback model in which Late cluster expression is a repression Hill function of Middle cluster expression levels and an activation Hill function of both RpaA ∼ P and RpaB ∼ P, presented as in ( C ). A model with an coherent feedforward architecture where the Middle cluster activates the Late cluster ( C ) best recapitulates the difference of the Late cluster responses to Shade and Clear Day Sunset. During the Shade pulse, the Middle cluster levels do not reach high enough levels to allow for strong activation of Late cluster expression, but at Sunset in Clear Day, Middle cluster levels reach high enough levels to activate the expression of the Late cluster.

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Expressing, Activation Assay, Produced

Journal: eLife

Article Title: Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

doi: 10.7554/eLife.32032

Figure Lengend Snippet:

Article Snippet: RpaA- and RpaB-conjugated Affigel 10/15 resin (Bio-Rad) was prepared following manufacturer’s instructions as described previously ( ).

Techniques: Recombinant, Plasmid Preparation, FLAG-tag, Selection, Marker, Transformation Assay, Western Blot, Produced, Affinity Purification, Software, Sample Prep