rabbit polyclonal anti gfp antibody  (Revvity Signals)

Journal: PLOS Biology

Article Title: Identification of a highly efficient chloroplast-targeting peptide for plastid engineering

doi: 10.1371/journal.pbio.3002785

Figure Lengend Snippet: ( A ) Amino acid sequences of selected cTPs whose import efficiencies into chloroplasts were re-assessed. The underlined amino acid sequences shown in magenta are the predicted cleavage sites within each cTP. The cTP score reports the predicted probability that these transit peptides are targeted to chloroplasts. ( B , C ) Distribution of GFP/chlorophyll fluorescence in plant cells and chloroplasts, presented as box plots ( n = 21, see numerical data in ). The black bars within the box plots represent the medians of the data distribution. Different letters indicate significant differences in the means, determined using one-way ANOVA with Tukey’s HSD test at p = 0.05. ( D ) CLSM images of tobacco leaf cells expressing different cTP-GFPs. Scale bars = 20 μm. ( E , F ) Immunoblotting of cTP-GFP in total leaf proteins and isolated chloroplast proteins, respectively. PS = Ponceau S staining, αRA1 = anti-Rubisco Activase 1 antibody, αGFP = anti-GFP antibody, RbcL = Rubisco-large subunit, and RCA1 = Rubisco activase 1. ( G ) Quantitative analysis of GFP levels in chloroplasts per in total leaf proteins. Error bars represent SD ( n = 3, ). Different letters indicate significant differences in the means, analyzed using one-way ANOVA with Tukey’s HSD test at p = 0.05. CLSM, confocal laser-scanning microscopy; cTP, chloroplast-targeting peptide; GFP, green fluorescent protein; SD, standard deviation.

Article Snippet: GFP bands on the membrane were probed with rabbit anti-GFP polyclonal antibodies (NB600-308; Novus Biologicals, Littleton, Colorado, USA) as the primary antibody at a dilution of 1:5,000.

Techniques: Fluorescence, Expressing, Western Blot, Isolation, Staining, Confocal Laser Scanning Microscopy, Standard Deviation

Journal: PLOS Biology

Article Title: Identification of a highly efficient chloroplast-targeting peptide for plastid engineering

doi: 10.1371/journal.pbio.3002785

Figure Lengend Snippet: ( A ) SDS-PAGE of various recombinant cTP-GFPs prepared using the E . coli expression system. One microgram of recombinant proteins was analyzed on a 14% SDS-PAGE gel before staining with CBB to visualize the protein bands. ( B ) Immunoblot analysis of recombinant cTP-GFPs expressed in cell-free protein synthesis reactions. Two microliters of each cell-free synthesis reaction was resolved by SDS-PAGE and immunoblotted using anti-GFP antibody. ( C , D ) Cleavage of recombinant At2g24090 cTP-GFP in E . coli cells. Cell lysates extracted from E . coli cultures with (+) and without (-) induction by IPTG were subjected to immunoblotting using anti-GFP (right panel in ( C )) and anti-His tag (right panel in ( D )) antibodies. The membranes were stained with CBB after immunoblotting (left panels in ( C , D )). ( E ) Immunoblot analysis of proteins with in vitro import activity. The abundance of different cTP-GFP precursors (Pre) and mature proteins (Mat) in import reactions collected at various time points post-incubation was analyzed by immunoblotting with anti-GFP antibody. The membranes were stained with CBB after immunoblotting to ensure equal loading of protein samples onto the membrane. ( F , G ) Import efficiencies of various recombinant cTP-GFPs into chloroplasts. The efficiencies of 3 selected cTPs for importing GFP into chloroplasts were determined via in vitro import assays as shown in ( E ). ( G ) Shows the linear regression of the import efficiencies of different cTP-GFPs at 0 to 15 min post-incubation. ( H ) Import constants of each cTP-GFP. Import constants for the recombinant cTP-GFPs were calculated using the linear regression equations of each protein at 2.5 to 15 min of incubation. Error bars in ( F – H ) represent the standard deviations of the mean from 3 biologically independent experiments ( n = 3). Different letters indicate significant differences among the recombinant proteins (one-way ANOVA with Tukey’s HSD test at p = 0.05). Quantitative data for Fig 3F–3H can be found in . CBB, Coomassie brilliant blue; cTP, chloroplast-targeting peptide; GFP, green fluorescent protein.

Article Snippet: GFP bands on the membrane were probed with rabbit anti-GFP polyclonal antibodies (NB600-308; Novus Biologicals, Littleton, Colorado, USA) as the primary antibody at a dilution of 1:5,000.

Techniques: SDS Page, Recombinant, Expressing, Staining, Western Blot, In Vitro, Activity Assay, Incubation, Membrane

Journal: PLOS Biology

Article Title: Identification of a highly efficient chloroplast-targeting peptide for plastid engineering

doi: 10.1371/journal.pbio.3002785

Figure Lengend Snippet: ( A ) Transcript expression of cTP-GFP genes in agroinfiltrated tobacco leaves at different time points post-agroinfiltration analyzed by qRT-PCR using GFP -specific primers. The expression levels of cTP-GFP transcripts were compared to that of pBI121-transformed leaves (EV) at 24 h post-infiltration. Relative transcript expression was determined from 3 biologically independent agroinfiltrated leaves ( n = 3, ). Error bars represent SD. ( B ) Abundance of various cTP-GFP fluorescent proteins in total leaf proteins. GFP fluorescence in total proteins extracted from agroinfiltrated tobacco leaves at different time points was measured, and the amounts of fluorescent protein in 3 experimentally independent samples ( n = 3, ) were calculated from the linear regression equations generated from the fluorescent intensities of standard GFP. Error bars represent SD. ( C ) Localization of various recombinant cTP-GFPs in transfected tobacco leaf cells at different time points post-agroinfiltration. Scale bars: 20 μm. ( D ) Ratio of GFP fluorescence in chloroplasts to total GFP fluorescence in a CLSM image. CLSM images were captured from transformed plant leaves at different time points. Fluorescent intensities of recombinant cTP-GFPs within chloroplasts and in an ROI of a CSLM image were measured using Fiji ImageJ. Error bars represent SD of 16 CLSM images ( n = 16, ) collected from 3 independent experiments. ( E ) Immunoblot analysis of recombinant cTP-GFPs in total leaf proteins. Total proteins from agroinfiltrated tobacco leaves at different time points post-infiltration were immunoblotted (WB) against anti-GFP polyclonal antibody to detect the recombinant cTP-GFP precursors (Pre) and the chloroplast-localized matured proteins (Mat) in samples. Equal loading of proteins onto the immunoblot membrane was confirmed by CBB staining of the Rubisco large subunit protein (RbcL) band. ( F , G ) Accumulation of various cTP-GFP precursors ( F ) and their matured proteins after translocation to chloroplasts ( G ). The content of recombinant proteins in total leaf proteins was determined from 3 biologically independent immunoblot experiments against the calibration equation of different amounts of standard GFP ( n = 3, ). Error bars represent SD. ( H ) Increase in matured GFP contents in total leaf proteins from 24 to 96 HAI. Error bars represent SD of the means of matured protein contents in total leaf proteins from 3 biologically independent samples ( n = 3, ). Asterisks in ( F ) to ( H ) indicate statistically significant differences analyzed by ANOVA with Dunnett’s multiple comparisons against the AtRbcS1A-GFP contents (*; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001, and ****; P < 0.0001). n.s. represent no significant difference. ( I ) Maturation rate constants of different cTP-GFP. Rate constants were determined from linear regression equations of matured protein contents in total leaf proteins at 24 to 48 HAI. Error bars represent SD of the average of rate constants from 3 experiments ( n = 3, ). Letters in ( I ) indicate different levels of statistically significant difference of means analyzed by one-way ANOVA with Tukey’s HSD test at p = 0.05. CBB, Coomassie brilliant blue; CLSM, confocal laser-scanning microscopy; cTP, chloroplast-targeting peptide; EV, empty vector; GFP, green fluorescent protein; HAI, hours after infiltration; qRT-PCR, quantitative reverse-transcription PCR; ROI, regions of interest; SD, standard deviation.

Article Snippet: GFP bands on the membrane were probed with rabbit anti-GFP polyclonal antibodies (NB600-308; Novus Biologicals, Littleton, Colorado, USA) as the primary antibody at a dilution of 1:5,000.

Techniques: Expressing, Quantitative RT-PCR, Transformation Assay, Fluorescence, Generated, Recombinant, Transfection, Western Blot, Membrane, Staining, Translocation Assay, Confocal Laser Scanning Microscopy, Plasmid Preparation, Reverse Transcription, Standard Deviation

Journal: PLOS Biology

Article Title: Identification of a highly efficient chloroplast-targeting peptide for plastid engineering

doi: 10.1371/journal.pbio.3002785

Figure Lengend Snippet: ( A ) Diagram illustrating recombinant truncated At2g24090 cTP-GFPs. ( B ) Immunoblot analysis of proteins recovered at 3 h after the import reaction of truncated At2g24090-GFPs and isolated tobacco chloroplasts using anti-GFP antibody. CBB staining shows equal protein loading. “Pre” indicates the precursor forms of recombinant At2g24090-GFPs; “Mat” represents matured (cleaved) cTP-GFPs. ( C ) Import efficiencies of At2g24090-GFP variants determined by immunoblotting as in ( B ). Error bars represent standard deviations of the mean from 3 independent experiments ( n = 3, ). Different letters indicate significant differences among treatments (one-way ANOVA with Tukey’s HSD test at p = 0.05). ( D ) Time-dependent import functions of truncated At2g24090-GFPs. Proteins extracted from import reactions at different time points (0–15 min) were analyzed by immunoblotting using anti-GFP antibody. CBB staining shows equal protein loading. ( E ) Import efficiencies of truncated At2g24090-GFPs. Chloroplast import efficiencies of recombinant proteins at different time points were determined by immunoblotting (as shown in ( D )). Error bars represent standard deviations of the mean from 3 assays ( n = 3, ). ( F ) Import constants of truncated At2g24090-GFPs into chloroplasts, calculated from the linear regression in ( E ) at time = 0 to 15 min (see numerical values in ). Error bars represent standard deviations. Asterisks indicate significant differences in the means of each treatment compared to 55-GFP (Student’s t test: *; p < 0.05, **; p < 0.01). n.s., no significant difference. ( G ) Subcellular localization of At2g24090-GFP variants in agroinfiltrated tobacco leaf cells at 3 DAI. Scale bars are 50 μm. ( H ) Immunoblot analysis of recombinant cTP-GFPs in total leaf proteins and isolated chloroplast proteins at 3 DAI using anti-GFP antibody to detect recombinant cTP-GFP levels in plant proteins prior to CBB staining. RbcL, large subunit of Rubisco. ( I ) Quantitative analysis of the import efficiencies of truncated At2g24090-GFPs in chloroplasts after agroinfiltration. The import efficiency of each cTP-GFP was determined by immunoblotting ( H ). Error bars represent standard deviations of the mean from 3 independent experiments ( n = 3, ). Asterisks indicate significant differences (Student’s t test: *; p < 0.05, **; p < 0.01, n.s., no significant difference). CBB, Coomassie brilliant blue; cTP, chloroplast-targeting peptide; GFP, green fluorescent protein.

Article Snippet: GFP bands on the membrane were probed with rabbit anti-GFP polyclonal antibodies (NB600-308; Novus Biologicals, Littleton, Colorado, USA) as the primary antibody at a dilution of 1:5,000.

Techniques: Recombinant, Western Blot, Isolation, Staining

Journal: PLOS Biology

Article Title: Identification of a highly efficient chloroplast-targeting peptide for plastid engineering

doi: 10.1371/journal.pbio.3002785

Figure Lengend Snippet: ( A ) Diagram of photosystems and electron transport complexes on the thylakoid membrane of the chloroplast. ( B ) RNA processing and activation of plastidial photosynthesis-related transcription by NtHCF152. ( C ) Expression cassettes of recombinant cTP-GFPs used to compare import efficiencies. ( D ) Localization of different recombinant cTP-GFPs within chloroplasts of transfected tobacco leaf cells after agroinfiltration for 3 DAI. Scale bars = 50 μm. ( E ) Comparative fluorescence analysis of recombinant cTP-GFPs within the chloroplasts of plant cells. Error bars represent standard deviations of the mean determined from 12 CLSM images from 2 independent experiments (see fluorescence values in ). Asterisks indicate a significant difference in the mean (Student’s t test). ( F , G ) Immunoblotting and quantitative analysis of the abundance of recombinant cTP-GFPs in total leaf proteins and chloroplast proteins. GFP-fused recombinant proteins expressed in plant cells and chloroplasts were analyzed using anti-GFP antibody. The band intensity of GFP-specific signal was quantified using Fiji ImageJ. Error bars in ( G ) represent the standard deviation of the mean from 4 independent transformation experiments ( n = 4, ). Different letters indicate significant differences in the mean among treatments (one-way ANOVA with Tukey’s HSD test at p = 0.05). ( H ) Expression constructs of cTP-engineered HCF152. EV = empty vector. ΔcTP = expression cassette for cTP-depleted NtHCF152 protein. ( I , J ) Expression of NtHCF152- and At2g24090 cTP-coding genes in tobacco leaves. ( K ) Transcript abundances of photosynthesis-related genes in tobacco leaves expressing recombinant At2g24090 cTP-HCF152 protein. Error bars represent the standard deviations of the mean from 5 transformation experiments ( n = 5). Different letters in ( I – K ) indicate significant differences in means (one-way ANOVA with Tukey’s HSD test at p = 0.05). Relative transcript expression values in Fig 7I–7K are available in . CLSM, confocal laser-scanning microscopy; cTP, chloroplast-targeting peptide; DAI, days after agroinfiltration; GFP, green fluorescent protein.

Article Snippet: GFP bands on the membrane were probed with rabbit anti-GFP polyclonal antibodies (NB600-308; Novus Biologicals, Littleton, Colorado, USA) as the primary antibody at a dilution of 1:5,000.

Techniques: Membrane, Activation Assay, Expressing, Recombinant, Transfection, Fluorescence, Western Blot, Standard Deviation, Transformation Assay, Construct, Plasmid Preparation, Confocal Laser Scanning Microscopy

Journal: bioRxiv

Article Title: PiggyBac mediated transgenesis and CRISPR/Cas9 knockout in the greater waxmoth, Galleria mellonella

doi: 10.1101/2024.09.17.613535

Figure Lengend Snippet: Constructs used to generate transgenic lines Gm hsp90:GFP-αtub1b and Bm hsp90:his2av-MCh (A). Gm hsp90:GFP-αtub1b (B, top left) with fat body tissue fixed and stained with an anti-GFP antibody (B, top right) and Bm hsp90:his2av-MCh (B, bottom left) larvae with fat body and dorsal neural ganglion imaged live (B, bottom left). Expected cystoskeletal distribution of eGFP was observed, corresponding with expected localisation of tubulin, with a nuclear localisation observed for mCherry. Brightfield (BF), mCherry and eGFP tissue expression patterns in a strain with both Bm hsp90:his2av-MCh/ Gm hsp90:GFP-αtub1b expression cassettes (C). Strong fat body expression was observed for both fluorophores, with the Galleria hsp90 promoter appears to drive strongest in gut and silk gland and the Bombyx hsp90 promoter stronger in epidermal and muscle tissue (not shown) but very weak in silk glands and malpighian tubules.

Article Snippet: Larval tissues were fixed in 4% paraformaldehyde/PBS + 0.1% Tween for 1 hour and stained overnight with a rabbit anti-GFP polyclonal antibody (Abcam 6556) and labelled using an appropriate Alexa Fluor 488 secondary dye (Molecular Probes).

Techniques: Construct, Transgenic Assay, Staining, Expressing