rnase t  (New England Biolabs)


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    Name:
    RNase B
    Description:
    RNase B 250 ug
    Catalog Number:
    p7817s
    Price:
    67
    Size:
    250 ug
    Category:
    Ribonucleases RNase
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    Structured Review

    New England Biolabs rnase t
    RNase B
    RNase B 250 ug
    https://www.bioz.com/result/rnase t/product/New England Biolabs
    Average 94 stars, based on 1040 article reviews
    Price from $9.99 to $1999.99
    rnase t - by Bioz Stars, 2021-02
    94/100 stars

    Images

    1) Product Images from "Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells"

    Article Title: Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2018.02.031

    Synthetic crRNAs with Terminal 2′-O-Me Nucleotides Can Mediate Gene Disruption in Human Cells  with the red arrow indicating the 5′ and 3′ ends of the crRNA (left). (Right) Schematic representation of scrRNAs with variable length direct repeat and DNA specificity region. 43-mer crRNA represents the natural crRNA of the AsCpf1 system. “Linker” nucleotides are underlined, and chemical substitutions are indicated by color as in (B). (D) Full sequence of DNMT1 targeting scrRNAs and single-experiment gene disruption activity compared with U-36-01 (normalized as 1). (E) SYBRgold-stained TBE-urea denaturing gel for visualization of RNA degradation products after a 30-min co-incubation with RNase T. (F) Gel same as in (E) except with the addition of multiple time points up to 60 min.
    Figure Legend Snippet: Synthetic crRNAs with Terminal 2′-O-Me Nucleotides Can Mediate Gene Disruption in Human Cells with the red arrow indicating the 5′ and 3′ ends of the crRNA (left). (Right) Schematic representation of scrRNAs with variable length direct repeat and DNA specificity region. 43-mer crRNA represents the natural crRNA of the AsCpf1 system. “Linker” nucleotides are underlined, and chemical substitutions are indicated by color as in (B). (D) Full sequence of DNMT1 targeting scrRNAs and single-experiment gene disruption activity compared with U-36-01 (normalized as 1). (E) SYBRgold-stained TBE-urea denaturing gel for visualization of RNA degradation products after a 30-min co-incubation with RNase T. (F) Gel same as in (E) except with the addition of multiple time points up to 60 min.

    Techniques Used: Sequencing, Activity Assay, Staining, Incubation

    2) Product Images from "Production of an Endo-?-N-Acetylglucosaminidase Activity Mediates Growth of Enterococcus faecalis on a High-Mannose-Type Glycoprotein"

    Article Title: Production of an Endo-?-N-Acetylglucosaminidase Activity Mediates Growth of Enterococcus faecalis on a High-Mannose-Type Glycoprotein

    Journal: Journal of Bacteriology

    doi:

    MALDI-TOF analysis of residual RNase B in culture supernatant during the growth of E. faecalis BC002. (a) Spectra of RNase B following 0, 1, 2, 3, and 4 h of incubation with E. faecalis (BC002). Culture supernatants were analyzed by MALDI-TOF mass spectrometry with sinapinic acid as the matrix. The 13,885- and 14,088-Da peaks correspond to the RNase B peptide backbone with one and two N -acetylglucosamine residues bound, respectively. (b) Relative proportions of RNase B glycoforms during the first 5 h of growth. Relative amounts of each glycoform were estimated from the peak height on MALDI-TOF spectra, with the 0-h amount taken as 100%. Amounts of Man 5 (⧫), Man 6 (■), Man 7 (▴), Man 8 , (●), and Man 9 (×) were measured.
    Figure Legend Snippet: MALDI-TOF analysis of residual RNase B in culture supernatant during the growth of E. faecalis BC002. (a) Spectra of RNase B following 0, 1, 2, 3, and 4 h of incubation with E. faecalis (BC002). Culture supernatants were analyzed by MALDI-TOF mass spectrometry with sinapinic acid as the matrix. The 13,885- and 14,088-Da peaks correspond to the RNase B peptide backbone with one and two N -acetylglucosamine residues bound, respectively. (b) Relative proportions of RNase B glycoforms during the first 5 h of growth. Relative amounts of each glycoform were estimated from the peak height on MALDI-TOF spectra, with the 0-h amount taken as 100%. Amounts of Man 5 (⧫), Man 6 (■), Man 7 (▴), Man 8 , (●), and Man 9 (×) were measured.

    Techniques Used: Incubation, Mass Spectrometry

    SDS-PAGE of residual RNase B during the growth of E. faecalis . Lanes 1 to 6, RNase B following 0, 1, 2, 3, 4, and 5 h of bacterial growth, respectively. Molecular mass markers are indicated by arrows.
    Figure Legend Snippet: SDS-PAGE of residual RNase B during the growth of E. faecalis . Lanes 1 to 6, RNase B following 0, 1, 2, 3, 4, and 5 h of bacterial growth, respectively. Molecular mass markers are indicated by arrows.

    Techniques Used: SDS Page

    Glycan structures present in the different glycoforms of RNase B. M, mannose; G,  N -acetylglucosamine; Asn, asparagine residue in the polypeptide. Mannose residues are linked via α(1→2), α(1→3), α(1→6), and β(1→4) linkages, which are indicated by the numbers 2, 3, 6, and 4, respectively.
    Figure Legend Snippet: Glycan structures present in the different glycoforms of RNase B. M, mannose; G, N -acetylglucosamine; Asn, asparagine residue in the polypeptide. Mannose residues are linked via α(1→2), α(1→3), α(1→6), and β(1→4) linkages, which are indicated by the numbers 2, 3, 6, and 4, respectively.

    Techniques Used:

    HPAEC analyses of free RNase B glycans present in the culture supernatant during growth of E. faecalis . (a) Glycans from heat-inactivated culture supernatant after 2 h of incubation were resolved by HPAEC using a gradient of 10 to 60 mM sodium acetate (0 to 30 min) in 100 mM sodium hydroxide. ∗, resolution of two Man 7 ). (b) Changes in the abundance of free glycans in the culture supernatant during exponential growth of E. faecalis as determined by HPAEC. Glycans were quantified by peak integration of detector response. Values for Man 5 -GlcNAc (⧫), Man 6 -GlcNAc (■), Man 7 -GlcNAc (▴), Man 8 -GlcNAc (●), and Man 9 -GlcNAc (×) are shown.
    Figure Legend Snippet: HPAEC analyses of free RNase B glycans present in the culture supernatant during growth of E. faecalis . (a) Glycans from heat-inactivated culture supernatant after 2 h of incubation were resolved by HPAEC using a gradient of 10 to 60 mM sodium acetate (0 to 30 min) in 100 mM sodium hydroxide. ∗, resolution of two Man 7 ). (b) Changes in the abundance of free glycans in the culture supernatant during exponential growth of E. faecalis as determined by HPAEC. Glycans were quantified by peak integration of detector response. Values for Man 5 -GlcNAc (⧫), Man 6 -GlcNAc (■), Man 7 -GlcNAc (▴), Man 8 -GlcNAc (●), and Man 9 -GlcNAc (×) are shown.

    Techniques Used: Incubation

    MALDI-TOF analysis of free RNase B glycans in the culture supernatant after 2 h of E. faecalis BC002 growth. The culture supernatant was subjected to reverse-phase chromatography to remove hydrophobic contaminants and was desalted by ion exchange. Samples were analyzed using 2,5-dihydroxybenzoic acid as the matrix. ∗, molecular ions plus phosphate (98 mass units).
    Figure Legend Snippet: MALDI-TOF analysis of free RNase B glycans in the culture supernatant after 2 h of E. faecalis BC002 growth. The culture supernatant was subjected to reverse-phase chromatography to remove hydrophobic contaminants and was desalted by ion exchange. Samples were analyzed using 2,5-dihydroxybenzoic acid as the matrix. ∗, molecular ions plus phosphate (98 mass units).

    Techniques Used: Reversed-phase Chromatography

    3) Product Images from "Characterizing the Release of Bioactive N-Glycans from Dairy Products by a Novel Endo-β-N-Acetylglucosaminidase"

    Article Title: Characterizing the Release of Bioactive N-Glycans from Dairy Products by a Novel Endo-β-N-Acetylglucosaminidase

    Journal: Biotechnology progress

    doi: 10.1002/btpr.2135

    Denaturation effect of concentrated bovine colostrum whey, bovine lactoferrin, and RNase B on N -glycan release by EndoBI-1 and PNGase F
    Figure Legend Snippet: Denaturation effect of concentrated bovine colostrum whey, bovine lactoferrin, and RNase B on N -glycan release by EndoBI-1 and PNGase F

    Techniques Used:

    Enzymatic deglycosylation of RNase B by Endo-B- N -acetylglucosaminidase and PNGase F on 12% SDS-PAGE gel. Lane 1: glycosylated RNase B (17kDa). Lane 2: deglycosylated RNase B by Endo- β-N -acetyl-glucosaminidase (47kDa). Lane 3: deglycosylated RNase
    Figure Legend Snippet: Enzymatic deglycosylation of RNase B by Endo-B- N -acetylglucosaminidase and PNGase F on 12% SDS-PAGE gel. Lane 1: glycosylated RNase B (17kDa). Lane 2: deglycosylated RNase B by Endo- β-N -acetyl-glucosaminidase (47kDa). Lane 3: deglycosylated RNase

    Techniques Used: SDS Page

    4) Product Images from "An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides"

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    Journal: Nature Communications

    doi: 10.1038/ncomms15487

    High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, Sequencing, Incubation, SDS Page

    The Fbs1 GYR variant substantially improves N-glycopeptide enrichment. The total ion chromatogram (TIC) (upper panel) from an LC-MS analysis shows wt Fbs1 or Fbs1 GYR-mediated binding and enrichment of N-glycopeptides from a complex peptide mixture. The complex peptide mixture is a tryptic digest of RNase B spiked with SGP and SGP-TMR. RNaseB contains non-glycosylated peptides and two major high-mannose N-glycopeptides labelled in the enlargement as: M5N2-NLTK and M6N2-NLTK. M5N2 or M6N2 indicates 5 or 6 mannose residues and 2 GlcNAc residues, respectively. NLTK is the peptide sequence of the N-glycopeptide produced by trypsin treatment of RNase B. The enrichment was performed in low salt (50 mM ammonium acetate, pH7.5). The black line indicates the chromatogram of a 50% input mixture. The orange and blue lines indicate the chromatograms of Fbs1 GYR and wt Fbs1 enrichment samples. The major N-glycopeptide peaks (M5N2-NLTK, M6N2-NLTK, SGP and SGP-TMR) are indicated. N-glycopeptides were quantified from the extracted ion chromatogram of the LC-MS analysis. The ions with the correct monoisotopic m/z values, that is, M5N2-NLTK:1691.98 1- (theoretical, 1691.72 1- ), M6N2-NLTK: 1854.07 1- (theoretical,1853.72 1- ) and SGP: 1433.47 2- (theoretical, 1433.10 2- ) were extracted, integrated and quantified. The amount of SGP-TMR was determined by fluorescence measurement of the LC elution. Recovery of each N-glycopeptide (enriched peptide amount/input peptide amount) is shown as a bar graph (lower panel). A representative TIC profile is shown from three experiments.
    Figure Legend Snippet: The Fbs1 GYR variant substantially improves N-glycopeptide enrichment. The total ion chromatogram (TIC) (upper panel) from an LC-MS analysis shows wt Fbs1 or Fbs1 GYR-mediated binding and enrichment of N-glycopeptides from a complex peptide mixture. The complex peptide mixture is a tryptic digest of RNase B spiked with SGP and SGP-TMR. RNaseB contains non-glycosylated peptides and two major high-mannose N-glycopeptides labelled in the enlargement as: M5N2-NLTK and M6N2-NLTK. M5N2 or M6N2 indicates 5 or 6 mannose residues and 2 GlcNAc residues, respectively. NLTK is the peptide sequence of the N-glycopeptide produced by trypsin treatment of RNase B. The enrichment was performed in low salt (50 mM ammonium acetate, pH7.5). The black line indicates the chromatogram of a 50% input mixture. The orange and blue lines indicate the chromatograms of Fbs1 GYR and wt Fbs1 enrichment samples. The major N-glycopeptide peaks (M5N2-NLTK, M6N2-NLTK, SGP and SGP-TMR) are indicated. N-glycopeptides were quantified from the extracted ion chromatogram of the LC-MS analysis. The ions with the correct monoisotopic m/z values, that is, M5N2-NLTK:1691.98 1- (theoretical, 1691.72 1- ), M6N2-NLTK: 1854.07 1- (theoretical,1853.72 1- ) and SGP: 1433.47 2- (theoretical, 1433.10 2- ) were extracted, integrated and quantified. The amount of SGP-TMR was determined by fluorescence measurement of the LC elution. Recovery of each N-glycopeptide (enriched peptide amount/input peptide amount) is shown as a bar graph (lower panel). A representative TIC profile is shown from three experiments.

    Techniques Used: Variant Assay, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Sequencing, Produced, Fluorescence

    Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, Variant Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Fluorescence

    Fbs1 binds to diverse types of N-glycomolecules. ( a ) Fbs1 binding to RNase B is N-glycan dependent. RNase B or deglycosylated RNase B was subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Lane 1, RNase B input control (CTL). Lane 2, essentially no RNase B deglycosylated by PNGase F is pulled down by Fbs1. Lane 3, RNase B with N-glycans is efficiently pulled down by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 binds to the N-glycosylated heavy chain of human IgG. Denatured and reduced human IgG were subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Heavy chains of human IgGs are typically N-glycosylated. Lane 1 is a control showing the IgG light chain and heavy chain. Lane 2 is Fbs1 beads only. Some SNAP-Fbs1 protein leaches from the prototype Fbs1 beads (denoted by an asterisk). Lanes 3 and 4 show that only the glycosylated heavy chain is bound by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( c ) Fbs1 binding affinity (Kd value) to sialylglycopeptide (SGP), M3N2 and M3N2F was measured by isothermal titration calorimetry. Structures of SGP, M3N2 and M3N2F are shown in the left panel. M3N2F is M3N2 with α1-6 fucosylation at the reducing end GlcNAc. The left panel summarizes the Kd values of SGP ( n =4), M3N2 ( n =5) and M3N2F ( n =5) interacting with wt Fbs1. There is no significant difference between the Kd values of M3N2 and M3N2F ( P value 0.85 > 0.05, mean±s.e.m., t -test, two-tailed). ( d ) wt Fbs1 shows binding bias to different N-glycopeptides. SGP was labelled with TMR fluorophore to facilitate detection. N-glycans of SGP-TMR (1) were then trimmed with exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Binding of the trimmed glycopeptides to Fbs1 beads was analysed. The relative binding affinity to wt Fbs1 is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is only indicated in N-glycopeptide structure 1. Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: Fbs1 binds to diverse types of N-glycomolecules. ( a ) Fbs1 binding to RNase B is N-glycan dependent. RNase B or deglycosylated RNase B was subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Lane 1, RNase B input control (CTL). Lane 2, essentially no RNase B deglycosylated by PNGase F is pulled down by Fbs1. Lane 3, RNase B with N-glycans is efficiently pulled down by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 binds to the N-glycosylated heavy chain of human IgG. Denatured and reduced human IgG were subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Heavy chains of human IgGs are typically N-glycosylated. Lane 1 is a control showing the IgG light chain and heavy chain. Lane 2 is Fbs1 beads only. Some SNAP-Fbs1 protein leaches from the prototype Fbs1 beads (denoted by an asterisk). Lanes 3 and 4 show that only the glycosylated heavy chain is bound by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( c ) Fbs1 binding affinity (Kd value) to sialylglycopeptide (SGP), M3N2 and M3N2F was measured by isothermal titration calorimetry. Structures of SGP, M3N2 and M3N2F are shown in the left panel. M3N2F is M3N2 with α1-6 fucosylation at the reducing end GlcNAc. The left panel summarizes the Kd values of SGP ( n =4), M3N2 ( n =5) and M3N2F ( n =5) interacting with wt Fbs1. There is no significant difference between the Kd values of M3N2 and M3N2F ( P value 0.85 > 0.05, mean±s.e.m., t -test, two-tailed). ( d ) wt Fbs1 shows binding bias to different N-glycopeptides. SGP was labelled with TMR fluorophore to facilitate detection. N-glycans of SGP-TMR (1) were then trimmed with exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Binding of the trimmed glycopeptides to Fbs1 beads was analysed. The relative binding affinity to wt Fbs1 is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is only indicated in N-glycopeptide structure 1. Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, SDS Page, CTL Assay, Isothermal Titration Calorimetry, Two Tailed Test, Fluorescence

    5) Product Images from "Substrate Binding and Active Site Residues in RNases E and G"

    Article Title: Substrate Binding and Active Site Residues in RNases E and G

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.063263

    A , schematic map of RNase G. This linear map of RNase G shows the domains inferred from alignment against RNase E. The boundaries are given by the residue numbers below the linear map ). The positions of mutations
    Figure Legend Snippet: A , schematic map of RNase G. This linear map of RNase G shows the domains inferred from alignment against RNase E. The boundaries are given by the residue numbers below the linear map ). The positions of mutations

    Techniques Used:

    Double filter assay of RNA binding by RNase G. ) was adapted for RNase G as described under “Experimental Procedures.” A typical set of data is shown. The left hand column in each panel contained no added
    Figure Legend Snippet: Double filter assay of RNA binding by RNase G. ) was adapted for RNase G as described under “Experimental Procedures.” A typical set of data is shown. The left hand column in each panel contained no added

    Techniques Used: RNA Binding Assay

    Fluorescence assay of RNase G. A , shows a  schematic  of RNA 1, a 108-nucleotide regulatory RNA that is a model substrate for RNase E. The major cleavage site is denoted by the  vertical arrow . BR14FD, a synthetic fluorescent substrate for RNase E or G is
    Figure Legend Snippet: Fluorescence assay of RNase G. A , shows a schematic of RNA 1, a 108-nucleotide regulatory RNA that is a model substrate for RNase E. The major cleavage site is denoted by the vertical arrow . BR14FD, a synthetic fluorescent substrate for RNase E or G is

    Techniques Used: Fluorescence

    6) Product Images from "Glycosylated Self-Assembled Monolayers for Arrays and Surface Analysis"

    Article Title: Glycosylated Self-Assembled Monolayers for Arrays and Surface Analysis

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-61779-373-8_6

    SPRi reflectivity image and the corresponding sensorgrams on a spotted glyco-SAM array composed of thiolated mannose, galactose and the glycoprotein RNase B. The bioactivity of immobilized glycans were verified via lectin binding with Con A (500 nM).
    Figure Legend Snippet: SPRi reflectivity image and the corresponding sensorgrams on a spotted glyco-SAM array composed of thiolated mannose, galactose and the glycoprotein RNase B. The bioactivity of immobilized glycans were verified via lectin binding with Con A (500 nM).

    Techniques Used: Binding Assay

    7) Product Images from "Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT"

    Article Title: Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT

    Journal:

    doi: 10.1038/sj.emboj.7600645

    Recognition of glycosylation mutants of RNase S-Protein by GT. ( A ) 35 S-labeled RNase B N34 (lanes 1–5) or RNase B N62 (lanes 6–10) was generated in vitro , and its S-Proteins prepared. Samples where incubated for 40 min with or without
    Figure Legend Snippet: Recognition of glycosylation mutants of RNase S-Protein by GT. ( A ) 35 S-labeled RNase B N34 (lanes 1–5) or RNase B N62 (lanes 6–10) was generated in vitro , and its S-Proteins prepared. Samples where incubated for 40 min with or without

    Techniques Used: Labeling, Generated, In Vitro, Incubation

    8) Product Images from "Circular RNA Is Expressed across the Eukaryotic Tree of Life"

    Article Title: Circular RNA Is Expressed across the Eukaryotic Tree of Life

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090859

    Circle-specific PCR and relative RNase R resistance. a) An example of circular and linear isoforms, in this case for the P. falciparum gene MAL13P1.337 , and circle- and linear-specific PCR design. PCR is performed on cDNA from total RNA that was mock-treated or RNase R-treated, or on P. falciparum genomic DNA. Circle-specific PCR amplifies from RNA but not genomic DNA; it amplifies the candidate junction (177 bp band) but also an unexpected band corresponding to a 4-1 circle. b) Quantitation of RNase R resistance. Plotted here is the relative RNase R resistance of a circular isoform compared to its counterpart linear isoform (ΔΔCt): RNase R resistance(circle) – RNase R resistance(linear); gray bars are standard errors. RNase R resistance (the log 2 fold-change in RNA isoform abundance with RNase R treatment) was measured by quantitative RT-PCR and taken as ΔCt = Ct(mock – treatment) – Ct(RNase R – treatment). RNase R resistance values for circular and linear isoforms are separately shown in Figure S1 . All linear isoforms were sensitive to RNase R, showing a greater than 32-fold drop in abundance after RNase R treatment (ΔCt
    Figure Legend Snippet: Circle-specific PCR and relative RNase R resistance. a) An example of circular and linear isoforms, in this case for the P. falciparum gene MAL13P1.337 , and circle- and linear-specific PCR design. PCR is performed on cDNA from total RNA that was mock-treated or RNase R-treated, or on P. falciparum genomic DNA. Circle-specific PCR amplifies from RNA but not genomic DNA; it amplifies the candidate junction (177 bp band) but also an unexpected band corresponding to a 4-1 circle. b) Quantitation of RNase R resistance. Plotted here is the relative RNase R resistance of a circular isoform compared to its counterpart linear isoform (ΔΔCt): RNase R resistance(circle) – RNase R resistance(linear); gray bars are standard errors. RNase R resistance (the log 2 fold-change in RNA isoform abundance with RNase R treatment) was measured by quantitative RT-PCR and taken as ΔCt = Ct(mock – treatment) – Ct(RNase R – treatment). RNase R resistance values for circular and linear isoforms are separately shown in Figure S1 . All linear isoforms were sensitive to RNase R, showing a greater than 32-fold drop in abundance after RNase R treatment (ΔCt

    Techniques Used: Polymerase Chain Reaction, Quantitation Assay, Quantitative RT-PCR

    9) Product Images from "Pervasive transcription fine-tunes replication origin activity"

    Article Title: Pervasive transcription fine-tunes replication origin activity

    Journal: eLife

    doi: 10.7554/eLife.40802

    Asymmetry of origin sensitivity to pervasive transcription. ( A ) Top: pervasive transcriptional landscape detected by RNAPII CRAC ( Candelli et al., 2018 ) at YLL026W ( HSP104 ) and ARS1206 in wild-type cells, both on Watson (blue) and Crick (red) strands, at 25°C (dark colors) and 37°C (light colors). The 5' ends and the sequences of the proposed primary ACS and the predicted secondary ACS for ARS1206 are shown. Bottom: schemes of the reporters containing the HSP104 gene and ARS1206 placed under the control of a doxycycline-repressible promoter ( P TETOFF ). The position of the amplicon used for the qPCR in ( B ) is shown. pS and pAS differ for the orientation of ARS1206 , with the primary (pS) or the secondary ACS (pAS) exposed to constitutive readthrough transcription from HSP104 . The sequence and the organization of the relevant region are indicated on the right for each plasmid. The positions of the oligonucleotides used for RNaseH cleavage (black arrows) and of the probe used in ( C ) are also indicated. The sequences of the oligonucleotides is reported in Table 1, with the following correspondence: cleaving oligo ‘a’=DL163; Northern probe = DL164; cleaving oligo ‘b’ = DL473; cleaving oligo ‘c’ = DL3991; cleaving oligo ‘d’ = DL3994. ( B ). Quantification by RT-qPCR of the HSP104 mRNA levels expressed from pS or pAS in the presence or absence of 5 µg/mL doxycycline. The position of the qPCR amplicon is reported in ( A ). ( C ). Northern blot analysis of HSP104 transcripts extracted from wild-type cells and subjected to RNAse H treatment before electrophoresis using oligonucleotides ‘a-d’ (positions shown in A ). All RNAs were cleaved with oligonucleotide ‘a’ to decrease the size of the fragments analyzed and detect small differences in size. Cleavage with oligonucleotide ‘b’ (oligo-dT) (lanes 3, 4) allowed erasing length heterogeneity due to poly(A) tails. Oligonucleotides ‘c’ and ‘d’ were added in reactions run in lanes 1 and 6, respectively, to detect possible longer products that might originate from significant levels of transcription readthrough from HSP104 , if the inversion of ARS1206 were to alter the transcription termination efficiency. Products of RNAse H degradation were run on a denaturing agarose gel and analyzed by Northern blot using a radiolabeled HSP104 probe (position shown in A ). ( D ). Stability of plasmids depending on ARS1206 for replication as a function of ARS orientation. pS or pAS was transformed in wild-type cells and single transformants were grown and maintained in logarythmic phase in YPD for several generations. To assess the loss of the transformed plasmid, cells were retrieved at the indicated number of generations and serial dilutions spotted on YPD (left) or minimal media lacking uracile (right) for 2 or 3 days, respectively, at 30°C. ( E ). Mutation of ORC2 affects more severely the stability of pAS compared to pS. Transformation of pS and pAS in wild-type ( ORC2 , ‘−‘) or mutant ( orc2-1 , ‘+') cells. Pictures were taken after 5 days of incubation at permissive temperature (23°C).
    Figure Legend Snippet: Asymmetry of origin sensitivity to pervasive transcription. ( A ) Top: pervasive transcriptional landscape detected by RNAPII CRAC ( Candelli et al., 2018 ) at YLL026W ( HSP104 ) and ARS1206 in wild-type cells, both on Watson (blue) and Crick (red) strands, at 25°C (dark colors) and 37°C (light colors). The 5' ends and the sequences of the proposed primary ACS and the predicted secondary ACS for ARS1206 are shown. Bottom: schemes of the reporters containing the HSP104 gene and ARS1206 placed under the control of a doxycycline-repressible promoter ( P TETOFF ). The position of the amplicon used for the qPCR in ( B ) is shown. pS and pAS differ for the orientation of ARS1206 , with the primary (pS) or the secondary ACS (pAS) exposed to constitutive readthrough transcription from HSP104 . The sequence and the organization of the relevant region are indicated on the right for each plasmid. The positions of the oligonucleotides used for RNaseH cleavage (black arrows) and of the probe used in ( C ) are also indicated. The sequences of the oligonucleotides is reported in Table 1, with the following correspondence: cleaving oligo ‘a’=DL163; Northern probe = DL164; cleaving oligo ‘b’ = DL473; cleaving oligo ‘c’ = DL3991; cleaving oligo ‘d’ = DL3994. ( B ). Quantification by RT-qPCR of the HSP104 mRNA levels expressed from pS or pAS in the presence or absence of 5 µg/mL doxycycline. The position of the qPCR amplicon is reported in ( A ). ( C ). Northern blot analysis of HSP104 transcripts extracted from wild-type cells and subjected to RNAse H treatment before electrophoresis using oligonucleotides ‘a-d’ (positions shown in A ). All RNAs were cleaved with oligonucleotide ‘a’ to decrease the size of the fragments analyzed and detect small differences in size. Cleavage with oligonucleotide ‘b’ (oligo-dT) (lanes 3, 4) allowed erasing length heterogeneity due to poly(A) tails. Oligonucleotides ‘c’ and ‘d’ were added in reactions run in lanes 1 and 6, respectively, to detect possible longer products that might originate from significant levels of transcription readthrough from HSP104 , if the inversion of ARS1206 were to alter the transcription termination efficiency. Products of RNAse H degradation were run on a denaturing agarose gel and analyzed by Northern blot using a radiolabeled HSP104 probe (position shown in A ). ( D ). Stability of plasmids depending on ARS1206 for replication as a function of ARS orientation. pS or pAS was transformed in wild-type cells and single transformants were grown and maintained in logarythmic phase in YPD for several generations. To assess the loss of the transformed plasmid, cells were retrieved at the indicated number of generations and serial dilutions spotted on YPD (left) or minimal media lacking uracile (right) for 2 or 3 days, respectively, at 30°C. ( E ). Mutation of ORC2 affects more severely the stability of pAS compared to pS. Transformation of pS and pAS in wild-type ( ORC2 , ‘−‘) or mutant ( orc2-1 , ‘+') cells. Pictures were taken after 5 days of incubation at permissive temperature (23°C).

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Northern Blot, Quantitative RT-PCR, Electrophoresis, Agarose Gel Electrophoresis, Transformation Assay, Mutagenesis, Incubation

    10) Product Images from "Identification of human genetic variants controlling circular RNA expression"

    Article Title: Identification of human genetic variants controlling circular RNA expression

    Journal: RNA

    doi: 10.1261/rna.071654.119

    .) ( A ) Divergent primers with respect to genomic sequence were designed for 17 circRNA candidates. These become properly inward-facing and identify the backsplice junction sequence. The left and right panels of the gel show human Jurkat and LCL cells, respectively. ( B ) Sanger sequencing confirms the backsplice junction sequence of the PCR products. Arrows indicate the presence of backsplice junction. ( C ) Divergent primers with respect to the genomic sequence amplify the circRNA backsplice junction sequence in the cDNA but not in the gDNA fraction. Convergent primers with respect to the genomic sequence amplify mRNA in both cDNA and gDNA fractions. ( D ) Treatment with RNase R leads to enrichment of circRNAs and depletion of the host gene and Beta-2 microglobulin (B2M) mRNAs compared to mock. ( E ) Average expression levels of the tested circRNAs in 358 EUR LCL samples of the 1000 Genomes Project RNA-seq data. Error bars indicate 95% confidence intervals.
    Figure Legend Snippet: .) ( A ) Divergent primers with respect to genomic sequence were designed for 17 circRNA candidates. These become properly inward-facing and identify the backsplice junction sequence. The left and right panels of the gel show human Jurkat and LCL cells, respectively. ( B ) Sanger sequencing confirms the backsplice junction sequence of the PCR products. Arrows indicate the presence of backsplice junction. ( C ) Divergent primers with respect to the genomic sequence amplify the circRNA backsplice junction sequence in the cDNA but not in the gDNA fraction. Convergent primers with respect to the genomic sequence amplify mRNA in both cDNA and gDNA fractions. ( D ) Treatment with RNase R leads to enrichment of circRNAs and depletion of the host gene and Beta-2 microglobulin (B2M) mRNAs compared to mock. ( E ) Average expression levels of the tested circRNAs in 358 EUR LCL samples of the 1000 Genomes Project RNA-seq data. Error bars indicate 95% confidence intervals.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Expressing, RNA Sequencing Assay

    11) Product Images from "Breaching self-tolerance to Alu duplex RNA underlies MDA5-mediated inflammation"

    Article Title: Breaching self-tolerance to Alu duplex RNA underlies MDA5-mediated inflammation

    Journal: Cell

    doi: 10.1016/j.cell.2017.12.016

    Paired Alus, not unpaired Alus, stimulate GOF MDA5 and are abundant in cytosol (A–C) Representative electron micrographs (A), IRF3 stimulatory activity (B), and ATPase activity (C) of G495R in complex with the sense (+) or antisense (−) strand of Alu from the NICN1 3’UTR. (D) Schematic of the RNase H-based method to selectively cleave unpaired, but not paired Alu RNAs. (E) Gel analysis of the RNase H assay. In vitro transcribed Alu RNAs (from NICN1 3’UTR) were subjected to the RNase H assay as described in (D). An oligo targeting GAPDH (αGAPDH) was used for negative controls. (F) Quantitation of Alu:Alu hybrids in cytosolic RNA. The RNase H assay in (D) was performed using purified cytosolic RNA from 293T cells, and remaining Alu(+) and Alu(−) were quantitated relative to the spike-in control. (G) The levels of Alu(+), Alu(−), GAPDH and ACTB (right) relative to the spike-in control before and after the RNase A protection assay. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5 and 2.0 ng/µl RNase A in the presence or absence of G495R. .
    Figure Legend Snippet: Paired Alus, not unpaired Alus, stimulate GOF MDA5 and are abundant in cytosol (A–C) Representative electron micrographs (A), IRF3 stimulatory activity (B), and ATPase activity (C) of G495R in complex with the sense (+) or antisense (−) strand of Alu from the NICN1 3’UTR. (D) Schematic of the RNase H-based method to selectively cleave unpaired, but not paired Alu RNAs. (E) Gel analysis of the RNase H assay. In vitro transcribed Alu RNAs (from NICN1 3’UTR) were subjected to the RNase H assay as described in (D). An oligo targeting GAPDH (αGAPDH) was used for negative controls. (F) Quantitation of Alu:Alu hybrids in cytosolic RNA. The RNase H assay in (D) was performed using purified cytosolic RNA from 293T cells, and remaining Alu(+) and Alu(−) were quantitated relative to the spike-in control. (G) The levels of Alu(+), Alu(−), GAPDH and ACTB (right) relative to the spike-in control before and after the RNase A protection assay. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5 and 2.0 ng/µl RNase A in the presence or absence of G495R. .

    Techniques Used: Activity Assay, Rnase H Assay, In Vitro, Quantitation Assay, Purification

    Alu:Alu hybrids formed by IR-Alus are the primary ligands for G495R MDA5 (A) Schematic of the RNase A protection assay. Cytosolic RNA (5 ng/µl) from 293T cells was pre-incubated with purified MDA5 G495R (150 nM), treated with RNase A, and recovered for subsequent biochemical and functional analyses (See Methods). (B) Western blot analysis of the 293T cytosolic fraction, from which cytosolic RNA was purified. (C and D) IRF3 dimerization (C), filament formation (D) assays with RNAs recovered from the G495R-protected digestion. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5, and 2.0 ng/µl of RNase A, respectively. Same mass concentrations (0.5 ng/µl for IRF3 dimerization and 2.0 ng/µl for EM) of RNAs were used. IRF3 dimerization was measured in the presence of an increasing concentration of competitor tRNA (cRNA, 0–8 ng/µl). (E) RNA-seq followed by RepeatMasker analysis of cytoRNA-0.0 and cytoRNA-2.0. The table below shows averages (standard deviations in parenthesis) of two independent biological repeats. (F) Normalized gene counts of cytoRNA-2.0 plotted against cytoRNA-0.0. (G) Distribution of sequencing reads of cytoRNA-0.0 and cytoRNA-2.0. Two representative genes ( BRI3BP and CXorf56 ) from the top enriched genes are shown. Thin, medium thick and thick lines represent intron, UTR and CDS, respectively, according to the GENCODE v24 annotation. Red arrows represent Alu elements according to the RepeatMasker annotation. Y-axis represents read count. (H) Schematic of Alus in the inverted repeat (IR) configuration. (I) Histograms of the enrichment factors of IR-Alus (gap between Alus
    Figure Legend Snippet: Alu:Alu hybrids formed by IR-Alus are the primary ligands for G495R MDA5 (A) Schematic of the RNase A protection assay. Cytosolic RNA (5 ng/µl) from 293T cells was pre-incubated with purified MDA5 G495R (150 nM), treated with RNase A, and recovered for subsequent biochemical and functional analyses (See Methods). (B) Western blot analysis of the 293T cytosolic fraction, from which cytosolic RNA was purified. (C and D) IRF3 dimerization (C), filament formation (D) assays with RNAs recovered from the G495R-protected digestion. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5, and 2.0 ng/µl of RNase A, respectively. Same mass concentrations (0.5 ng/µl for IRF3 dimerization and 2.0 ng/µl for EM) of RNAs were used. IRF3 dimerization was measured in the presence of an increasing concentration of competitor tRNA (cRNA, 0–8 ng/µl). (E) RNA-seq followed by RepeatMasker analysis of cytoRNA-0.0 and cytoRNA-2.0. The table below shows averages (standard deviations in parenthesis) of two independent biological repeats. (F) Normalized gene counts of cytoRNA-2.0 plotted against cytoRNA-0.0. (G) Distribution of sequencing reads of cytoRNA-0.0 and cytoRNA-2.0. Two representative genes ( BRI3BP and CXorf56 ) from the top enriched genes are shown. Thin, medium thick and thick lines represent intron, UTR and CDS, respectively, according to the GENCODE v24 annotation. Red arrows represent Alu elements according to the RepeatMasker annotation. Y-axis represents read count. (H) Schematic of Alus in the inverted repeat (IR) configuration. (I) Histograms of the enrichment factors of IR-Alus (gap between Alus

    Techniques Used: Incubation, Purification, Functional Assay, Western Blot, Concentration Assay, RNA Sequencing Assay, Sequencing

    12) Product Images from "Brain clusterin protein isoforms and mitochondrial localization"

    Article Title: Brain clusterin protein isoforms and mitochondrial localization

    Journal: eLife

    doi: 10.7554/eLife.48255

    Positive controls for deglycosylation studies. RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates.
    Figure Legend Snippet: Positive controls for deglycosylation studies. RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates.

    Techniques Used: Positive Control

    13) Product Images from "An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide"

    Article Title: An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide

    Journal: Scientific Reports

    doi: 10.1038/srep13370

    Def N-terminus is not modified by glycosylation. ( a ) Diagram showing different Def derivatives. Numeration denotes the position of amino acid residue. Number of amino acid residues in each peptide is shown in brackets. ( b ) Western blot using anti-EGFP antibody to detect EGFP-D14 and EGFP-D15 (left panel). Right panel shows the ΔMW (MW SDS−PAGE − MW predicted ) for EGFP-D14 and EGFP-D15. ( c ) Western blot using an EGFP antibody to detect EGFP-D14 and EGFP-D15 treated with or without PNGase or O-glycosidase plus Neuraminidase. Proteins samples were extracted from embryos at 8 hpf after EGFP-D14 or EGFP-D15 mRNA injection into one-cell stage embryos. ( d ) Rnase B and Fetuin were used as the positive controls in glycosidase treatment as indicated and were stained with CBB (coomassie brilliant blue). The gel picture (for CBB staining) and western blot images were cropped with a grey cropping line. All gels for western blot analysis were run under the same experimental conditions.
    Figure Legend Snippet: Def N-terminus is not modified by glycosylation. ( a ) Diagram showing different Def derivatives. Numeration denotes the position of amino acid residue. Number of amino acid residues in each peptide is shown in brackets. ( b ) Western blot using anti-EGFP antibody to detect EGFP-D14 and EGFP-D15 (left panel). Right panel shows the ΔMW (MW SDS−PAGE − MW predicted ) for EGFP-D14 and EGFP-D15. ( c ) Western blot using an EGFP antibody to detect EGFP-D14 and EGFP-D15 treated with or without PNGase or O-glycosidase plus Neuraminidase. Proteins samples were extracted from embryos at 8 hpf after EGFP-D14 or EGFP-D15 mRNA injection into one-cell stage embryos. ( d ) Rnase B and Fetuin were used as the positive controls in glycosidase treatment as indicated and were stained with CBB (coomassie brilliant blue). The gel picture (for CBB staining) and western blot images were cropped with a grey cropping line. All gels for western blot analysis were run under the same experimental conditions.

    Techniques Used: Modification, Western Blot, SDS Page, Injection, Staining

    14) Product Images from "The pCri System: A Vector Collection for Recombinant Protein Expression and Purification"

    Article Title: The pCri System: A Vector Collection for Recombinant Protein Expression and Purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112643

    Protein expression and purification trials using the pCri System. ( A ) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in  E. coli  BL21 cells, and subsequently purified by Ni-NTA-affinity chromatography except for MBP, GST, and LSL fusion products, which were purified by their respective specific affinity resins. ( B ) The gene coding for fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in  E. coli  Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. All expression trials were performed at 20°C except for pCri-1a, which was also performed at 37°C. ( C ) Partially purified MBP-fragilysin before (−) and after (+) TEV proteinase cleavage. Arrows indicate the soluble fraction of fragilysin (white) and the MBP (black) after TEV proteinase cleavage. ( D ) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in  E. coli  cells, and extracellularly (lanes 5 and 6) in  P. pastoris  cells. Lanes indicate samples before (1, 3 and 5) and after (2, 4 and 6) tryptic digestion. Arrows indicate the pro-CPA2 (black), the mature form (grey) and the pro-peptide (white) after tryptic cleavage. ( E ) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in  E. coli  BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. ( F ) Activity of affinity-purified TRX-PNGase F against glycosylated RNase B. (+) and (−) indicate presence and absence of PNGase F. Arrows indicate the PNGase F (black), native RNase B (grey) and deglycosylated RNase B (white). ( G ) MecR1 was expressed in  E. coli  BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “  Materials and Methods ”. A black arrow indicates the detected MecR1. ( H ) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. ( I ) Partially purified MBP-GFP, SUMO-GFP and MISTIC-MecR1 were digested with TEV proteinase, SENP1 or thrombin, respectively. For TEV proteinase and SENP1 digestions various ratios of proteinase∶tagged-protein were tested in overnight incubations at 4°C, whereas for thrombin digestions 2 units of proteinase were used to digest 25 µg of protein for various times at room temperature. Arrows indicate tagged-protein (black), target protein (grey) and fused-tag (white) after proteinase cleavage.
    Figure Legend Snippet: Protein expression and purification trials using the pCri System. ( A ) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in E. coli BL21 cells, and subsequently purified by Ni-NTA-affinity chromatography except for MBP, GST, and LSL fusion products, which were purified by their respective specific affinity resins. ( B ) The gene coding for fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in E. coli Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. All expression trials were performed at 20°C except for pCri-1a, which was also performed at 37°C. ( C ) Partially purified MBP-fragilysin before (−) and after (+) TEV proteinase cleavage. Arrows indicate the soluble fraction of fragilysin (white) and the MBP (black) after TEV proteinase cleavage. ( D ) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in E. coli cells, and extracellularly (lanes 5 and 6) in P. pastoris cells. Lanes indicate samples before (1, 3 and 5) and after (2, 4 and 6) tryptic digestion. Arrows indicate the pro-CPA2 (black), the mature form (grey) and the pro-peptide (white) after tryptic cleavage. ( E ) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in E. coli BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. ( F ) Activity of affinity-purified TRX-PNGase F against glycosylated RNase B. (+) and (−) indicate presence and absence of PNGase F. Arrows indicate the PNGase F (black), native RNase B (grey) and deglycosylated RNase B (white). ( G ) MecR1 was expressed in E. coli BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “ Materials and Methods ”. A black arrow indicates the detected MecR1. ( H ) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. ( I ) Partially purified MBP-GFP, SUMO-GFP and MISTIC-MecR1 were digested with TEV proteinase, SENP1 or thrombin, respectively. For TEV proteinase and SENP1 digestions various ratios of proteinase∶tagged-protein were tested in overnight incubations at 4°C, whereas for thrombin digestions 2 units of proteinase were used to digest 25 µg of protein for various times at room temperature. Arrows indicate tagged-protein (black), target protein (grey) and fused-tag (white) after proteinase cleavage.

    Techniques Used: Expressing, Purification, Clone Assay, Affinity Chromatography, SDS Page, Activity Assay, Affinity Purification, Western Blot

    15) Product Images from "Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus"

    Article Title: Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus

    Journal: Nature Communications

    doi: 10.1038/s41467-019-10246-5

    Biological functions of circE7 in tumors and episomal HPV. CaSki cells (4×10 6 ), which had been stably transduced with the indicated construct, were xenografted onto the flanks of NSG mice ( n = 8 per construct). Mice were given water with or without doxycycline (1 mg/mL) as indicated. a Image of representative CaSki tumor xenografts dissected from the indicated mice after 21 days (top). Weights of CaSki tumors with or without dox-induced circE7 sh1/2 expression (bottom). b Representative images of tumors formed by CaSki xenografts without (top) or with (bottom) doxycycline. Arrowhead indicates an area of invasive tumor. Arrows indicate mitotic figures and Ki-67-positive cells. Dashed box indicates area of detail. Scale bars, 200 μm. c TCGA RNA-Seq data (CESC, HNSC) was analyzed with vircircRNA and backsplices with ≥2 reads were tabulated. d RT-PCR from CaSki or HPV BP cells that possess integrated or episomal HPV16 genomes with or without RNase R reveals the presence of circE7 in both samples. e Human foreskin keratinocyte (HFK), keratinocytes infected with religated HPV31 (HFK + HPV31), or a HPV31 infected cell line derived from a grade II cervical biopsy (CIN612) were induced to differentiate with high calcium. Levels of HPV31 circE7 were assessed by RT-PCR (left) or RT-qPCR (right). Calcium-induced differentiation significantly decreased levels of HPV31 circE7. RT-PCR is representative of 4 independent experiments. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( a , e )
    Figure Legend Snippet: Biological functions of circE7 in tumors and episomal HPV. CaSki cells (4×10 6 ), which had been stably transduced with the indicated construct, were xenografted onto the flanks of NSG mice ( n = 8 per construct). Mice were given water with or without doxycycline (1 mg/mL) as indicated. a Image of representative CaSki tumor xenografts dissected from the indicated mice after 21 days (top). Weights of CaSki tumors with or without dox-induced circE7 sh1/2 expression (bottom). b Representative images of tumors formed by CaSki xenografts without (top) or with (bottom) doxycycline. Arrowhead indicates an area of invasive tumor. Arrows indicate mitotic figures and Ki-67-positive cells. Dashed box indicates area of detail. Scale bars, 200 μm. c TCGA RNA-Seq data (CESC, HNSC) was analyzed with vircircRNA and backsplices with ≥2 reads were tabulated. d RT-PCR from CaSki or HPV BP cells that possess integrated or episomal HPV16 genomes with or without RNase R reveals the presence of circE7 in both samples. e Human foreskin keratinocyte (HFK), keratinocytes infected with religated HPV31 (HFK + HPV31), or a HPV31 infected cell line derived from a grade II cervical biopsy (CIN612) were induced to differentiate with high calcium. Levels of HPV31 circE7 were assessed by RT-PCR (left) or RT-qPCR (right). Calcium-induced differentiation significantly decreased levels of HPV31 circE7. RT-PCR is representative of 4 independent experiments. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( a , e )

    Techniques Used: Stable Transfection, Transduction, Construct, Mouse Assay, Expressing, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Infection, Derivative Assay, Quantitative RT-PCR, Two Tailed Test

    Identification of HPV circRNAs. a A transcript map generated by vircircRNA summarizing the splicing events identified for HPV16 from the combined SRA datasets (Supplementary Fig. 1e ). Lines (top) indicate forward splicing events; arcs (bottom) indicate backsplicing; thickness = log 2 (read count); red arc highlights circE7. The lower panel represents a partial HPV16 genome with promoters (P, green arrowheads) and the early polyadenylation (A E , red line) indicated. Numbering from the NC_001526 reference sequence. b Alignment of sequencing reads spanning the circE7 backsplice junction from SRS2410540. Red indicates E7-E1 sequences, and blue indicates E6 sequence. c Predicted formation and size of HPV16 circE7. Arrows indicate primers used to detect linear E6/E7 and circE7. d RT-PCR of random hexamer primed total RNA from HPV16+ cancer cell lines. 2 μg of total RNA were treated with 5U of RNase R (or water for mock) in the presence of RNase inhibitor for 40 min prior to RT reaction. Results are representative of 4 independent experiments. e Sanger sequencing of PCR products from d confirmed the presence of the expected circE7 backsplice junction without the insertion of additional nucleotides. Sequencing traces were identical for 3 independent reactions from each cell line. f Northern blot of total RNA after mock (8 μg) or with RNase R treatment (20 μg) from the indicated HPV16+ cell line probed with HPV16 E7. Arrows indicates RNase resistant band with E7 sequence. Ethidium Bromide staining (bottom), RNase R treatment control. Results representative of 5 independent northerns
    Figure Legend Snippet: Identification of HPV circRNAs. a A transcript map generated by vircircRNA summarizing the splicing events identified for HPV16 from the combined SRA datasets (Supplementary Fig. 1e ). Lines (top) indicate forward splicing events; arcs (bottom) indicate backsplicing; thickness = log 2 (read count); red arc highlights circE7. The lower panel represents a partial HPV16 genome with promoters (P, green arrowheads) and the early polyadenylation (A E , red line) indicated. Numbering from the NC_001526 reference sequence. b Alignment of sequencing reads spanning the circE7 backsplice junction from SRS2410540. Red indicates E7-E1 sequences, and blue indicates E6 sequence. c Predicted formation and size of HPV16 circE7. Arrows indicate primers used to detect linear E6/E7 and circE7. d RT-PCR of random hexamer primed total RNA from HPV16+ cancer cell lines. 2 μg of total RNA were treated with 5U of RNase R (or water for mock) in the presence of RNase inhibitor for 40 min prior to RT reaction. Results are representative of 4 independent experiments. e Sanger sequencing of PCR products from d confirmed the presence of the expected circE7 backsplice junction without the insertion of additional nucleotides. Sequencing traces were identical for 3 independent reactions from each cell line. f Northern blot of total RNA after mock (8 μg) or with RNase R treatment (20 μg) from the indicated HPV16+ cell line probed with HPV16 E7. Arrows indicates RNase resistant band with E7 sequence. Ethidium Bromide staining (bottom), RNase R treatment control. Results representative of 5 independent northerns

    Techniques Used: Generated, Sequencing, Reverse Transcription Polymerase Chain Reaction, Random Hexamer Labeling, Polymerase Chain Reaction, Northern Blot, Staining

    Generation and translation of circE7.  a  Diagram of constructs generated to express circE7 in vitro. Map indicates location of QKI sites, start codons (all mutated in ‘noATG’), 3xFLAG (present in ‘FLAG’), splice sites (mutated in ‘SASD’), and siRNA used in subsequent experiments.  b  RT-PCR confirms the formation of RNase R-resistant circE7 RNAs from transfected 293 T cells. Results representative of  > 6 independent experiments.  c  Western blot for FLAG from 293 T cells co-transfected with circE7_FLAG and the indicated siRNA. GAPDH, loading control. Results representative of 4 independent transfections.  d  Western blots for FLAG and HPV16 E7 from 293 T cells transfected with the indicated circE7_FLAG construct (4 μg) or a linear FLAG-GFP control vector (0.4 μg). Indicated transfections were subjected to a heat shock (2 h at 42 °C, 2 h recovery). Eight-fold less lysate was loaded for control FLAG-GFP transfected cells. GAPDH, loading control. Results representative of 5 independent experiments.  e  RT-PCR demonstrates that splice sites indicated in  a  are required for the formation of circE7 RNAs.  f  RT-qPCR from 293 T cells transfected with pcDNA3.1-circE7 constructs confirms significantly lower circRNA formation in the SASD mutants. Results representative of 3 independent experiments.  g  Western blots for HPV16 E7 from 293 T cells transfected with the indicated circE7 construct (4 μg). GAPDH, loading control. Source data for  b ,  d ,  e , and  g  provided in Source Data file
    Figure Legend Snippet: Generation and translation of circE7. a Diagram of constructs generated to express circE7 in vitro. Map indicates location of QKI sites, start codons (all mutated in ‘noATG’), 3xFLAG (present in ‘FLAG’), splice sites (mutated in ‘SASD’), and siRNA used in subsequent experiments. b RT-PCR confirms the formation of RNase R-resistant circE7 RNAs from transfected 293 T cells. Results representative of  > 6 independent experiments. c Western blot for FLAG from 293 T cells co-transfected with circE7_FLAG and the indicated siRNA. GAPDH, loading control. Results representative of 4 independent transfections. d Western blots for FLAG and HPV16 E7 from 293 T cells transfected with the indicated circE7_FLAG construct (4 μg) or a linear FLAG-GFP control vector (0.4 μg). Indicated transfections were subjected to a heat shock (2 h at 42 °C, 2 h recovery). Eight-fold less lysate was loaded for control FLAG-GFP transfected cells. GAPDH, loading control. Results representative of 5 independent experiments. e RT-PCR demonstrates that splice sites indicated in a are required for the formation of circE7 RNAs. f RT-qPCR from 293 T cells transfected with pcDNA3.1-circE7 constructs confirms significantly lower circRNA formation in the SASD mutants. Results representative of 3 independent experiments. g Western blots for HPV16 E7 from 293 T cells transfected with the indicated circE7 construct (4 μg). GAPDH, loading control. Source data for b , d , e , and g provided in Source Data file

    Techniques Used: Construct, Generated, In Vitro, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Plasmid Preparation, Quantitative RT-PCR

    Protein encoding circE7 is essential for CaSki cell growth. a CaSki cells were lentivirally transduced with doxycycline (dox)-inducible hairpins specific for the circE7 backsplice junction (circE7 sh1/2). RT-qPCR for levels of circE7 revealed that circE7 sh1/2 resulted in significant decreases of circE7 levels. ( n = 3 independent experiments, run in duplicate). b Northern blot of RNase R treated total RNA (30 μg) from CaSki cells with or without circE7 sh1/2 induction (2 days). Band density (bottom number) was quantitated and normalized to the uninduced control. c Western blots for E7 and E6 after circE7 sh1/2 induction (3 days). Western blots representative of 3 independent experiments. GAPDH, loading control. d A total of 6.0 × 10 4 CaSki cells were seeded in triplicate in six-well plates at day 0 and absolute cell number quantitated daily after day 2. CircE7 sh1/2 induction resulted in significantly slower growth of CaSki cells after day 4. Similar results were obtained in 3 independent experiments. e CaSki cells with or without circE7 sh1/2 induction (1 day) were plated in chamber slides and labeled with BrdU (10 μM for 1.5 h). Cells were stained with αBrdU and DAPI and scored as % of DAPI + cells. f 1.0 × 10 4 CaSki circE7 sh1/2 cells with or without induction (1 day) were seeded in triplicate in soft agar with or without dox (14 days). Average colonies per 35 mm. n = 4 independent transfections. g CaSki were doubly transduced with a shRNA resistant WT circE7 (circResist_WT) and circE7 sh1/2. MTT assay of circResist_WT cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. h CaSki were doubly transduced with a shRNA resistant circE7 with no start codons (circResist_noATG) and circE7 sh1/2. MTT assay of circResist_noATG cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( d , g , h ) and one-way analysis of variance (ANOVA) with Holm–Sidak tests ( a , e , f ). Source data for b , c provided in Source Data file
    Figure Legend Snippet: Protein encoding circE7 is essential for CaSki cell growth. a CaSki cells were lentivirally transduced with doxycycline (dox)-inducible hairpins specific for the circE7 backsplice junction (circE7 sh1/2). RT-qPCR for levels of circE7 revealed that circE7 sh1/2 resulted in significant decreases of circE7 levels. ( n = 3 independent experiments, run in duplicate). b Northern blot of RNase R treated total RNA (30 μg) from CaSki cells with or without circE7 sh1/2 induction (2 days). Band density (bottom number) was quantitated and normalized to the uninduced control. c Western blots for E7 and E6 after circE7 sh1/2 induction (3 days). Western blots representative of 3 independent experiments. GAPDH, loading control. d A total of 6.0 × 10 4 CaSki cells were seeded in triplicate in six-well plates at day 0 and absolute cell number quantitated daily after day 2. CircE7 sh1/2 induction resulted in significantly slower growth of CaSki cells after day 4. Similar results were obtained in 3 independent experiments. e CaSki cells with or without circE7 sh1/2 induction (1 day) were plated in chamber slides and labeled with BrdU (10 μM for 1.5 h). Cells were stained with αBrdU and DAPI and scored as % of DAPI + cells. f 1.0 × 10 4 CaSki circE7 sh1/2 cells with or without induction (1 day) were seeded in triplicate in soft agar with or without dox (14 days). Average colonies per 35 mm. n = 4 independent transfections. g CaSki were doubly transduced with a shRNA resistant WT circE7 (circResist_WT) and circE7 sh1/2. MTT assay of circResist_WT cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. h CaSki were doubly transduced with a shRNA resistant circE7 with no start codons (circResist_noATG) and circE7 sh1/2. MTT assay of circResist_noATG cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( d , g , h ) and one-way analysis of variance (ANOVA) with Holm–Sidak tests ( a , e , f ). Source data for b , c provided in Source Data file

    Techniques Used: Transduction, Quantitative RT-PCR, Northern Blot, Western Blot, Labeling, Staining, Transfection, shRNA, MTT Assay, Two Tailed Test

    Characterization of circE7. a CircE7-transfected cells were fractionated and indicated fractions analyzed by northern blot. Total RNA (4 μg) with mock or RNase R treatment of fractions from 293 T cells confirms that circE7 is enriched in the cytoplasm and is RNase R-resistant. MALAT1 and β-actin, fractionation controls. Band density (bottom) quantitated after normalization to the enriched fraction. Results are representative of 3 independent blots. b CircE7-transfected 293T (left) or untransfected CaSki (right) were fractionated and analyzed by RT-qPCR. MALAT1 and 18 S (top), fractionation controls. Values normalized to the enriched fraction. Results are representative of 3 independent fractionation experiments. c RT-qPCR of RNA IP (m 6 A or IgG control) after transfection with the indicated plasmid (24 h) ( n = 8 biological replicates from 4 transfections). SON, m 6 A RNA IP control. d Western blot for METTL3 from 293T co-transfected with control or METTL3 siRNA and circE7 construct (top). GAPDH, loading control. RT-qPCR of RNA IP (m 6 A or IgG control) from 293 T cells co-transfected with indicated siRNA and circE7 construct. RT-PCR is representative of 4 independent experiments. e Schematic of the DRACH consensus motifs for METTL3/14 and the sites mutated in the circE7_noDRACH construct (top). RT-qPCR for circE7 in cells transfected with the indicated construct. Loss of UTR DRACH motifs in circE7 results in a significant decrease in the abundance of circE7, but not linear E6/E7. ( n = 4 independent experiments). f Western blot for E7 from 293 T transfected with indicated circE7 construct. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with one-way analysis of variance (ANOVA) with Holm–Sidak tests. g Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated (left). Dashed lines indicate collected fraction. Detection of circE7 in polysome fraction by RT-PCR after transfection with circE7 or circE7_noATG (right). β-actin, control. Source data for a provided in Source Data file
    Figure Legend Snippet: Characterization of circE7. a CircE7-transfected cells were fractionated and indicated fractions analyzed by northern blot. Total RNA (4 μg) with mock or RNase R treatment of fractions from 293 T cells confirms that circE7 is enriched in the cytoplasm and is RNase R-resistant. MALAT1 and β-actin, fractionation controls. Band density (bottom) quantitated after normalization to the enriched fraction. Results are representative of 3 independent blots. b CircE7-transfected 293T (left) or untransfected CaSki (right) were fractionated and analyzed by RT-qPCR. MALAT1 and 18 S (top), fractionation controls. Values normalized to the enriched fraction. Results are representative of 3 independent fractionation experiments. c RT-qPCR of RNA IP (m 6 A or IgG control) after transfection with the indicated plasmid (24 h) ( n = 8 biological replicates from 4 transfections). SON, m 6 A RNA IP control. d Western blot for METTL3 from 293T co-transfected with control or METTL3 siRNA and circE7 construct (top). GAPDH, loading control. RT-qPCR of RNA IP (m 6 A or IgG control) from 293 T cells co-transfected with indicated siRNA and circE7 construct. RT-PCR is representative of 4 independent experiments. e Schematic of the DRACH consensus motifs for METTL3/14 and the sites mutated in the circE7_noDRACH construct (top). RT-qPCR for circE7 in cells transfected with the indicated construct. Loss of UTR DRACH motifs in circE7 results in a significant decrease in the abundance of circE7, but not linear E6/E7. ( n = 4 independent experiments). f Western blot for E7 from 293 T transfected with indicated circE7 construct. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with one-way analysis of variance (ANOVA) with Holm–Sidak tests. g Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated (left). Dashed lines indicate collected fraction. Detection of circE7 in polysome fraction by RT-PCR after transfection with circE7 or circE7_noATG (right). β-actin, control. Source data for a provided in Source Data file

    Techniques Used: Transfection, Northern Blot, Fractionation, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Construct, Reverse Transcription Polymerase Chain Reaction

    16) Product Images from "CircRNA_005647通过结合miR-27b-3p抑制小鼠心肌成纤维细胞中纤维化相关基因表达"

    Article Title: CircRNA_005647通过结合miR-27b-3p抑制小鼠心肌成纤维细胞中纤维化相关基因表达

    Journal: Journal of Southern Medical University

    doi: 10.12122/j.issn.1673-4254.2019.11.08

    CircRNA_005647在心肌成纤维细胞中的表达、分布和稳定性鉴定 Expression, cellular distribution and stability of circRNA_005647 in mouse cardiac fibroblasts (CFs). A :Protein expression of fibrosis-related genes in Ang-Ⅱ-treated CFs; B :Expression of fibrosis-related genes, circRNA_005647 and Myo9a in Ang-Ⅱ-treated CFs by RT-qPCR assay; C :RT-qPCR showing abundant circRNA_005647 in the cytoplasm of the CFs; D :Resistance of circRNA_005647 against RNase R treatment by RT-qPCR assay; E :Detection of circRNA_005647 and Myo9a mRNA in the CFs after actinomycin D treatment. * P
    Figure Legend Snippet: CircRNA_005647在心肌成纤维细胞中的表达、分布和稳定性鉴定 Expression, cellular distribution and stability of circRNA_005647 in mouse cardiac fibroblasts (CFs). A :Protein expression of fibrosis-related genes in Ang-Ⅱ-treated CFs; B :Expression of fibrosis-related genes, circRNA_005647 and Myo9a in Ang-Ⅱ-treated CFs by RT-qPCR assay; C :RT-qPCR showing abundant circRNA_005647 in the cytoplasm of the CFs; D :Resistance of circRNA_005647 against RNase R treatment by RT-qPCR assay; E :Detection of circRNA_005647 and Myo9a mRNA in the CFs after actinomycin D treatment. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    17) Product Images from "Rotavirus VP3 targets MAVS for degradation to inhibit type III interferon expression in intestinal epithelial cells"

    Article Title: Rotavirus VP3 targets MAVS for degradation to inhibit type III interferon expression in intestinal epithelial cells

    Journal: eLife

    doi: 10.7554/eLife.39494

    SPLTSS phosphorylation mediates MAVS degradation by RV infection. ( A ) HEK293 cells were transfected with indicated Flag-tagged MAVS mutants for 48 hr with or without human RV Wa infection (MOI = 3) for the last 12 hr. The levels of MAVS and GAPDH were measured by western blot. ( B ) Purified recombinant Flag-MAVS and RNaseB proteins were digested with PNGaseF or EndoH and measured by silver staining (upper panel) and western blot for potential AMPylation (lower panel). Top arrow marks recombinant MAVS protein (~72 kD) and bottom arrow marks recombinant RNaseB protein (~17 kD). ( C ) HEK293 cells were transfected with indicated Flag-tagged MAVS mutants (SPLTSS: SPLTSS mutated to six alanines; R3A: R232, R236, R239 mutated to three alanines) for 48 hr with or without human RV Wa infection (MOI = 3) for the last 12 hr. The levels of MAVS and GAPDH were measured by western blot. ( D ) MAVS -/- HEK293 cells were transfected with WT or indicated MAVS mutants for 48 hr and harvested for RT-qPCR analysis measuring IFN-β and IFN-λ expression. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: SPLTSS phosphorylation mediates MAVS degradation by RV infection. ( A ) HEK293 cells were transfected with indicated Flag-tagged MAVS mutants for 48 hr with or without human RV Wa infection (MOI = 3) for the last 12 hr. The levels of MAVS and GAPDH were measured by western blot. ( B ) Purified recombinant Flag-MAVS and RNaseB proteins were digested with PNGaseF or EndoH and measured by silver staining (upper panel) and western blot for potential AMPylation (lower panel). Top arrow marks recombinant MAVS protein (~72 kD) and bottom arrow marks recombinant RNaseB protein (~17 kD). ( C ) HEK293 cells were transfected with indicated Flag-tagged MAVS mutants (SPLTSS: SPLTSS mutated to six alanines; R3A: R232, R236, R239 mutated to three alanines) for 48 hr with or without human RV Wa infection (MOI = 3) for the last 12 hr. The levels of MAVS and GAPDH were measured by western blot. ( D ) MAVS -/- HEK293 cells were transfected with WT or indicated MAVS mutants for 48 hr and harvested for RT-qPCR analysis measuring IFN-β and IFN-λ expression. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection, Transfection, Western Blot, Purification, Recombinant, Silver Staining, Quantitative RT-PCR, Expressing

    18) Product Images from "Substrate Binding and Active Site Residues in RNases E and G"

    Article Title: Substrate Binding and Active Site Residues in RNases E and G

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.063263

    A , schematic map of RNase G. This linear map of RNase G shows the domains inferred from alignment against RNase E. The boundaries are given by the residue numbers below the linear map ). The positions of mutations
    Figure Legend Snippet: A , schematic map of RNase G. This linear map of RNase G shows the domains inferred from alignment against RNase E. The boundaries are given by the residue numbers below the linear map ). The positions of mutations

    Techniques Used:

    Double filter assay of RNA binding by RNase G. ) was adapted for RNase G as described under “Experimental Procedures.” A typical set of data is shown. The left hand column in each panel contained no added
    Figure Legend Snippet: Double filter assay of RNA binding by RNase G. ) was adapted for RNase G as described under “Experimental Procedures.” A typical set of data is shown. The left hand column in each panel contained no added

    Techniques Used: RNA Binding Assay

    Fluorescence assay of RNase G. A , shows a  schematic  of RNA 1, a 108-nucleotide regulatory RNA that is a model substrate for RNase E. The major cleavage site is denoted by the  vertical arrow . BR14FD, a synthetic fluorescent substrate for RNase E or G is
    Figure Legend Snippet: Fluorescence assay of RNase G. A , shows a schematic of RNA 1, a 108-nucleotide regulatory RNA that is a model substrate for RNase E. The major cleavage site is denoted by the vertical arrow . BR14FD, a synthetic fluorescent substrate for RNase E or G is

    Techniques Used: Fluorescence

    19) Product Images from "Glycosylated Self-Assembled Monolayers for Arrays and Surface Analysis"

    Article Title: Glycosylated Self-Assembled Monolayers for Arrays and Surface Analysis

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-61779-373-8_6

    SPRi reflectivity image and the corresponding sensorgrams on a spotted glyco-SAM array composed of thiolated mannose, galactose and the glycoprotein RNase B. The bioactivity of immobilized glycans were verified via lectin binding with Con A (500 nM).
    Figure Legend Snippet: SPRi reflectivity image and the corresponding sensorgrams on a spotted glyco-SAM array composed of thiolated mannose, galactose and the glycoprotein RNase B. The bioactivity of immobilized glycans were verified via lectin binding with Con A (500 nM).

    Techniques Used: Binding Assay

    20) Product Images from "Brain clusterin protein isoforms and mitochondrial localization"

    Article Title: Brain clusterin protein isoforms and mitochondrial localization

    Journal: eLife

    doi: 10.7554/eLife.48255

    Positive controls for deglycosylation studies. RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates.
    Figure Legend Snippet: Positive controls for deglycosylation studies. RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates.

    Techniques Used: Positive Control

    21) Product Images from "Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size 1"

    Article Title: Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size 1

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.115.137687

    Deglycosylation of FOLR2. PNGase F treatment (500 U per reaction) was used to determine whether N-glycosylation affected the apparent size of FOLR2 detected in mouse placenta and P15 growing oocytes. Numbers at right indicate calculated molecular weights. The activity of PNGase F was confirmed using RNAse B detected with Ponceau S staining (top), showing the expected decrease in apparent size from 17 to 14 kDa. The added PNGase F was visible as a band at 36 kDa. Western blot analysis of the same membrane for FOLR2 (middle) did not detect any shift in the FOLR2 band (27 kDa) in mouse placenta lysate (10 μg/lane). Overexpressed human FOLR2 (200 ng), however, was decreased from 33 to 25 kDa upon deglycosylation. P15 oocyte (50 oocytes per lane) FOLR2 appeared to undergo a small decrease from about 31 to 30 kDa upon treatment with PNGase F. The β-actin served as a loading control (bottom). The example shown is representative of four independent repeats.
    Figure Legend Snippet: Deglycosylation of FOLR2. PNGase F treatment (500 U per reaction) was used to determine whether N-glycosylation affected the apparent size of FOLR2 detected in mouse placenta and P15 growing oocytes. Numbers at right indicate calculated molecular weights. The activity of PNGase F was confirmed using RNAse B detected with Ponceau S staining (top), showing the expected decrease in apparent size from 17 to 14 kDa. The added PNGase F was visible as a band at 36 kDa. Western blot analysis of the same membrane for FOLR2 (middle) did not detect any shift in the FOLR2 band (27 kDa) in mouse placenta lysate (10 μg/lane). Overexpressed human FOLR2 (200 ng), however, was decreased from 33 to 25 kDa upon deglycosylation. P15 oocyte (50 oocytes per lane) FOLR2 appeared to undergo a small decrease from about 31 to 30 kDa upon treatment with PNGase F. The β-actin served as a loading control (bottom). The example shown is representative of four independent repeats.

    Techniques Used: Activity Assay, Staining, Western Blot

    22) Product Images from "Biofunctional Paper via Covalent Modification of Cellulose"

    Article Title: Biofunctional Paper via Covalent Modification of Cellulose

    Journal: Langmuir : the ACS journal of surfaces and colloids

    doi: 10.1021/la301661x

    (a) DVS-activated membranes were stored for 60 days and subsequently functionalized with mannose, glucose, maltose, and RNase B. The bioactivity, as observed by fluorescent ConA detection, was minimally affected by storage of the DVS-activated paper.
    Figure Legend Snippet: (a) DVS-activated membranes were stored for 60 days and subsequently functionalized with mannose, glucose, maltose, and RNase B. The bioactivity, as observed by fluorescent ConA detection, was minimally affected by storage of the DVS-activated paper.

    Techniques Used:

    23) Product Images from "Glycomic analysis using glycoprotein immobilization for glycan extraction"

    Article Title: Glycomic analysis using glycoprotein immobilization for glycan extraction

    Journal: Analytical chemistry

    doi: 10.1021/ac400761e

    Coupling of protein onto the solid support. Protein, ribonuclease B (RNase B, 400 µg), was mixed with 200 µL of Aminolink slurry after denatured in 100°C for 10 min. Protein concentration was determined after conjugation protein
    Figure Legend Snippet: Coupling of protein onto the solid support. Protein, ribonuclease B (RNase B, 400 µg), was mixed with 200 µL of Aminolink slurry after denatured in 100°C for 10 min. Protein concentration was determined after conjugation protein

    Techniques Used: Protein Concentration, Conjugation Assay

    24) Product Images from "High-Level Expression of Endo-β-N-Acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and Its Application for the Deglycosylation of Glycoproteins"

    Article Title: High-Level Expression of Endo-β-N-Acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and Its Application for the Deglycosylation of Glycoproteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120458

    Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured RNase B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.
    Figure Legend Snippet: Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured RNase B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.

    Techniques Used: Activity Assay, Mobility Shift, Molecular Weight, Negative Control, Positive Control

    Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B. (A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.
    Figure Legend Snippet: Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B. (A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.

    Techniques Used: Mobility Shift, SDS Page, Molecular Weight, Negative Control, Positive Control

    25) Product Images from "Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells"

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2019.00176

    qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using ProtoScript ®  first strand cDNA synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH.  ∗∗  and  ∗∗∗  denote  p  ≤ 0.01 and  p  ≤ 0.001, respectively.  (A)  HeLa cells;  (B)  MCF and Jurkat cells.
    Figure Legend Snippet: qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using ProtoScript ® first strand cDNA synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

    Validation of candidate circRNA by cloning. HeLa cells were treated with cisplatin in three biological replicates as explained in Materials and Methods. Total RNAs from each replicate of control DMSO (1.1–1.3) and cisplatin (2.1–2.3) were run on 1% agarose gel for visual analyses (A) . Total RNAs were treated with RNAse R to eliminate linear RNAs followed by reverse transcription and PCR amplification. Lanes 1, 2, 5, and 6 (B,C) are the PCR products from hsa_circ_0012992, hsa_circ_HIPK3, hsa_circ_0014824, and hsa_circ_0029693, respectively. The efficiency of RNAse R treatment was measured by PCR-amplifying the linear beta-actin mRNA before (Lane 4) and after (Lane 3) the RNAse treatment. The gel-purified PCR fragments were cloned into the pCR ® II TA vector (Thermo Fisher Scientific, Unite States) and sequenced to identify the backsplice junction.
    Figure Legend Snippet: Validation of candidate circRNA by cloning. HeLa cells were treated with cisplatin in three biological replicates as explained in Materials and Methods. Total RNAs from each replicate of control DMSO (1.1–1.3) and cisplatin (2.1–2.3) were run on 1% agarose gel for visual analyses (A) . Total RNAs were treated with RNAse R to eliminate linear RNAs followed by reverse transcription and PCR amplification. Lanes 1, 2, 5, and 6 (B,C) are the PCR products from hsa_circ_0012992, hsa_circ_HIPK3, hsa_circ_0014824, and hsa_circ_0029693, respectively. The efficiency of RNAse R treatment was measured by PCR-amplifying the linear beta-actin mRNA before (Lane 4) and after (Lane 3) the RNAse treatment. The gel-purified PCR fragments were cloned into the pCR ® II TA vector (Thermo Fisher Scientific, Unite States) and sequenced to identify the backsplice junction.

    Techniques Used: Clone Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Purification, Plasmid Preparation

    Analyses of expression levels of circular and their counterpart linear mRNAs. For circRNA analyses, experiments were carried out as explained in Figure 3 . For mRNA analyses, RNAse R treatment was omitted before cDNA synthesis to amplify the linear RNAs. (A) HeLa cells treated with cisplatin only; (B) HeLa cells treated with cisplatin, doxorubicin, anti Fas, and TNF-alpha. Data, collected in three biological replicates, were normalized against DMSO control and GAPDH. ∗ , ∗∗ , and ∗∗∗ denote p ≤ 0.5, p ≤ 0.01, and p ≤ 0.001, respectively.
    Figure Legend Snippet: Analyses of expression levels of circular and their counterpart linear mRNAs. For circRNA analyses, experiments were carried out as explained in Figure 3 . For mRNA analyses, RNAse R treatment was omitted before cDNA synthesis to amplify the linear RNAs. (A) HeLa cells treated with cisplatin only; (B) HeLa cells treated with cisplatin, doxorubicin, anti Fas, and TNF-alpha. Data, collected in three biological replicates, were normalized against DMSO control and GAPDH. ∗ , ∗∗ , and ∗∗∗ denote p ≤ 0.5, p ≤ 0.01, and p ≤ 0.001, respectively.

    Techniques Used: Expressing

    Related Articles

    Incubation:

    Article Title: Pervasive transcription fine-tunes replication origin activity
    Article Snippet: .. RNase H cleavage was performed by annealing 50pmoles of each oligonucleotide to 20 µg of total RNAs in 1X RNase H buffer (NEB) followed by addition of 2U of RNase H (NEB) and incubation at 30°C for 45 min. ..

    Article Title: Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT
    Article Snippet: .. A 10 mg portion of RNase B was incubated with 5 μl endo Hf (NEB) in NEB-buffer G5 for 16 h at 37°C. endo Hf was removed by gel filtration via Sephadex75. .. Preparative treatment with α-mannosidase to generate the Man1 GlcNAc2 glycoform was performed in 50 mM citrate buffer pH 4.4 for 16 h at 37°C using 0.5 U α-mannosidase (Sigma) for 10 mg RNase B.

    Binding Assay:

    Article Title: Substrate Binding and Active Site Residues in RNases E and G
    Article Snippet: .. We adapted the double filter assay of Wong and Lohman ( ) to measure the binding of 5′-32 P-Ribo-8 to RNase G. Briefly, incubations containing 20 n m RNA oligonucleotide and from 100 n m to 4 m m RNase G were assembled on ice in FAB buffer (20 m m PIPES, 50 m m NaCl, 0.5 m m EDTA, 0.5 m m dithiothreitol, 20 μg/ml acetylated bovine serum albumin (New England Biolabs), pH 6.5). .. Samples were applied to individual wells in a Bio-Rad 96 well dot-blot apparatus containing a Bio-Rad nitrocellulose membrane over an Amersham Biosciences Hybond-NX nylon membrane, both previously washed with 1× FAB at room temperature.

    Filtration:

    Article Title: Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT
    Article Snippet: .. A 10 mg portion of RNase B was incubated with 5 μl endo Hf (NEB) in NEB-buffer G5 for 16 h at 37°C. endo Hf was removed by gel filtration via Sephadex75. .. Preparative treatment with α-mannosidase to generate the Man1 GlcNAc2 glycoform was performed in 50 mM citrate buffer pH 4.4 for 16 h at 37°C using 0.5 U α-mannosidase (Sigma) for 10 mg RNase B.

    other:

    Article Title: Glycosylated Self-Assembled Monolayers for Arrays and Surface Analysis
    Article Snippet: RNase B: 10 mg RNase B (New England Biolabs, Ipswich, MA), 10× PBS, pH 7.4.

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    New England Biolabs rnase t
    Synthetic crRNAs with Terminal 2′-O-Me Nucleotides Can Mediate Gene Disruption in Human Cells  with the red arrow indicating the 5′ and 3′ ends of the crRNA (left). (Right) Schematic representation of scrRNAs with variable length direct repeat and DNA specificity region. 43-mer crRNA represents the natural crRNA of the AsCpf1 system. “Linker” nucleotides are underlined, and chemical substitutions are indicated by color as in (B). (D) Full sequence of DNMT1 targeting scrRNAs and single-experiment gene disruption activity compared with U-36-01 (normalized as 1). (E) SYBRgold-stained TBE-urea denaturing gel for visualization of RNA degradation products after a 30-min co-incubation with RNase T. (F) Gel same as in (E) except with the addition of multiple time points up to 60 min.
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    Synthetic crRNAs with Terminal 2′-O-Me Nucleotides Can Mediate Gene Disruption in Human Cells  with the red arrow indicating the 5′ and 3′ ends of the crRNA (left). (Right) Schematic representation of scrRNAs with variable length direct repeat and DNA specificity region. 43-mer crRNA represents the natural crRNA of the AsCpf1 system. “Linker” nucleotides are underlined, and chemical substitutions are indicated by color as in (B). (D) Full sequence of DNMT1 targeting scrRNAs and single-experiment gene disruption activity compared with U-36-01 (normalized as 1). (E) SYBRgold-stained TBE-urea denaturing gel for visualization of RNA degradation products after a 30-min co-incubation with RNase T. (F) Gel same as in (E) except with the addition of multiple time points up to 60 min.

    Journal: Molecular Therapy

    Article Title: Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells

    doi: 10.1016/j.ymthe.2018.02.031

    Figure Lengend Snippet: Synthetic crRNAs with Terminal 2′-O-Me Nucleotides Can Mediate Gene Disruption in Human Cells with the red arrow indicating the 5′ and 3′ ends of the crRNA (left). (Right) Schematic representation of scrRNAs with variable length direct repeat and DNA specificity region. 43-mer crRNA represents the natural crRNA of the AsCpf1 system. “Linker” nucleotides are underlined, and chemical substitutions are indicated by color as in (B). (D) Full sequence of DNMT1 targeting scrRNAs and single-experiment gene disruption activity compared with U-36-01 (normalized as 1). (E) SYBRgold-stained TBE-urea denaturing gel for visualization of RNA degradation products after a 30-min co-incubation with RNase T. (F) Gel same as in (E) except with the addition of multiple time points up to 60 min.

    Article Snippet: 3 U of RNase T (NEB) was added to each reaction, and 7 μL was removed at corresponding time points, added to 2X TBE-UREA sample buffer (Thermo Fisher Scientific), and heated to 95°C for 10 min.

    Techniques: Sequencing, Activity Assay, Staining, Incubation

    MALDI-TOF analysis of residual RNase B in culture supernatant during the growth of E. faecalis BC002. (a) Spectra of RNase B following 0, 1, 2, 3, and 4 h of incubation with E. faecalis (BC002). Culture supernatants were analyzed by MALDI-TOF mass spectrometry with sinapinic acid as the matrix. The 13,885- and 14,088-Da peaks correspond to the RNase B peptide backbone with one and two N -acetylglucosamine residues bound, respectively. (b) Relative proportions of RNase B glycoforms during the first 5 h of growth. Relative amounts of each glycoform were estimated from the peak height on MALDI-TOF spectra, with the 0-h amount taken as 100%. Amounts of Man 5 (⧫), Man 6 (■), Man 7 (▴), Man 8 , (●), and Man 9 (×) were measured.

    Journal: Journal of Bacteriology

    Article Title: Production of an Endo-?-N-Acetylglucosaminidase Activity Mediates Growth of Enterococcus faecalis on a High-Mannose-Type Glycoprotein

    doi:

    Figure Lengend Snippet: MALDI-TOF analysis of residual RNase B in culture supernatant during the growth of E. faecalis BC002. (a) Spectra of RNase B following 0, 1, 2, 3, and 4 h of incubation with E. faecalis (BC002). Culture supernatants were analyzed by MALDI-TOF mass spectrometry with sinapinic acid as the matrix. The 13,885- and 14,088-Da peaks correspond to the RNase B peptide backbone with one and two N -acetylglucosamine residues bound, respectively. (b) Relative proportions of RNase B glycoforms during the first 5 h of growth. Relative amounts of each glycoform were estimated from the peak height on MALDI-TOF spectra, with the 0-h amount taken as 100%. Amounts of Man 5 (⧫), Man 6 (■), Man 7 (▴), Man 8 , (●), and Man 9 (×) were measured.

    Article Snippet: Glycans were also released from intact RNase B by treatment with either endo-β- N -acetylglucosaminidase H (Endo H) or peptide- N -glycosidase F (PNGase F) purchased from New England Biolabs Ltd. (Hitchin, Hertfordshire, United Kingdom).

    Techniques: Incubation, Mass Spectrometry

    SDS-PAGE of residual RNase B during the growth of E. faecalis . Lanes 1 to 6, RNase B following 0, 1, 2, 3, 4, and 5 h of bacterial growth, respectively. Molecular mass markers are indicated by arrows.

    Journal: Journal of Bacteriology

    Article Title: Production of an Endo-?-N-Acetylglucosaminidase Activity Mediates Growth of Enterococcus faecalis on a High-Mannose-Type Glycoprotein

    doi:

    Figure Lengend Snippet: SDS-PAGE of residual RNase B during the growth of E. faecalis . Lanes 1 to 6, RNase B following 0, 1, 2, 3, 4, and 5 h of bacterial growth, respectively. Molecular mass markers are indicated by arrows.

    Article Snippet: Glycans were also released from intact RNase B by treatment with either endo-β- N -acetylglucosaminidase H (Endo H) or peptide- N -glycosidase F (PNGase F) purchased from New England Biolabs Ltd. (Hitchin, Hertfordshire, United Kingdom).

    Techniques: SDS Page

    Glycan structures present in the different glycoforms of RNase B. M, mannose; G,  N -acetylglucosamine; Asn, asparagine residue in the polypeptide. Mannose residues are linked via α(1→2), α(1→3), α(1→6), and β(1→4) linkages, which are indicated by the numbers 2, 3, 6, and 4, respectively.

    Journal: Journal of Bacteriology

    Article Title: Production of an Endo-?-N-Acetylglucosaminidase Activity Mediates Growth of Enterococcus faecalis on a High-Mannose-Type Glycoprotein

    doi:

    Figure Lengend Snippet: Glycan structures present in the different glycoforms of RNase B. M, mannose; G, N -acetylglucosamine; Asn, asparagine residue in the polypeptide. Mannose residues are linked via α(1→2), α(1→3), α(1→6), and β(1→4) linkages, which are indicated by the numbers 2, 3, 6, and 4, respectively.

    Article Snippet: Glycans were also released from intact RNase B by treatment with either endo-β- N -acetylglucosaminidase H (Endo H) or peptide- N -glycosidase F (PNGase F) purchased from New England Biolabs Ltd. (Hitchin, Hertfordshire, United Kingdom).

    Techniques:

    HPAEC analyses of free RNase B glycans present in the culture supernatant during growth of E. faecalis . (a) Glycans from heat-inactivated culture supernatant after 2 h of incubation were resolved by HPAEC using a gradient of 10 to 60 mM sodium acetate (0 to 30 min) in 100 mM sodium hydroxide. ∗, resolution of two Man 7 ). (b) Changes in the abundance of free glycans in the culture supernatant during exponential growth of E. faecalis as determined by HPAEC. Glycans were quantified by peak integration of detector response. Values for Man 5 -GlcNAc (⧫), Man 6 -GlcNAc (■), Man 7 -GlcNAc (▴), Man 8 -GlcNAc (●), and Man 9 -GlcNAc (×) are shown.

    Journal: Journal of Bacteriology

    Article Title: Production of an Endo-?-N-Acetylglucosaminidase Activity Mediates Growth of Enterococcus faecalis on a High-Mannose-Type Glycoprotein

    doi:

    Figure Lengend Snippet: HPAEC analyses of free RNase B glycans present in the culture supernatant during growth of E. faecalis . (a) Glycans from heat-inactivated culture supernatant after 2 h of incubation were resolved by HPAEC using a gradient of 10 to 60 mM sodium acetate (0 to 30 min) in 100 mM sodium hydroxide. ∗, resolution of two Man 7 ). (b) Changes in the abundance of free glycans in the culture supernatant during exponential growth of E. faecalis as determined by HPAEC. Glycans were quantified by peak integration of detector response. Values for Man 5 -GlcNAc (⧫), Man 6 -GlcNAc (■), Man 7 -GlcNAc (▴), Man 8 -GlcNAc (●), and Man 9 -GlcNAc (×) are shown.

    Article Snippet: Glycans were also released from intact RNase B by treatment with either endo-β- N -acetylglucosaminidase H (Endo H) or peptide- N -glycosidase F (PNGase F) purchased from New England Biolabs Ltd. (Hitchin, Hertfordshire, United Kingdom).

    Techniques: Incubation

    MALDI-TOF analysis of free RNase B glycans in the culture supernatant after 2 h of E. faecalis BC002 growth. The culture supernatant was subjected to reverse-phase chromatography to remove hydrophobic contaminants and was desalted by ion exchange. Samples were analyzed using 2,5-dihydroxybenzoic acid as the matrix. ∗, molecular ions plus phosphate (98 mass units).

    Journal: Journal of Bacteriology

    Article Title: Production of an Endo-?-N-Acetylglucosaminidase Activity Mediates Growth of Enterococcus faecalis on a High-Mannose-Type Glycoprotein

    doi:

    Figure Lengend Snippet: MALDI-TOF analysis of free RNase B glycans in the culture supernatant after 2 h of E. faecalis BC002 growth. The culture supernatant was subjected to reverse-phase chromatography to remove hydrophobic contaminants and was desalted by ion exchange. Samples were analyzed using 2,5-dihydroxybenzoic acid as the matrix. ∗, molecular ions plus phosphate (98 mass units).

    Article Snippet: Glycans were also released from intact RNase B by treatment with either endo-β- N -acetylglucosaminidase H (Endo H) or peptide- N -glycosidase F (PNGase F) purchased from New England Biolabs Ltd. (Hitchin, Hertfordshire, United Kingdom).

    Techniques: Reversed-phase Chromatography

    Denaturation effect of concentrated bovine colostrum whey, bovine lactoferrin, and RNase B on N -glycan release by EndoBI-1 and PNGase F

    Journal: Biotechnology progress

    Article Title: Characterizing the Release of Bioactive N-Glycans from Dairy Products by a Novel Endo-β-N-Acetylglucosaminidase

    doi: 10.1002/btpr.2135

    Figure Lengend Snippet: Denaturation effect of concentrated bovine colostrum whey, bovine lactoferrin, and RNase B on N -glycan release by EndoBI-1 and PNGase F

    Article Snippet: Two microliters of 500,000 units/mL PNGase F were added to 20 μ g of RNase B reconstituted in 20 μ L of commercial denaturing buffer (New England BioLabs).

    Techniques:

    Enzymatic deglycosylation of RNase B by Endo-B- N -acetylglucosaminidase and PNGase F on 12% SDS-PAGE gel. Lane 1: glycosylated RNase B (17kDa). Lane 2: deglycosylated RNase B by Endo- β-N -acetyl-glucosaminidase (47kDa). Lane 3: deglycosylated RNase

    Journal: Biotechnology progress

    Article Title: Characterizing the Release of Bioactive N-Glycans from Dairy Products by a Novel Endo-β-N-Acetylglucosaminidase

    doi: 10.1002/btpr.2135

    Figure Lengend Snippet: Enzymatic deglycosylation of RNase B by Endo-B- N -acetylglucosaminidase and PNGase F on 12% SDS-PAGE gel. Lane 1: glycosylated RNase B (17kDa). Lane 2: deglycosylated RNase B by Endo- β-N -acetyl-glucosaminidase (47kDa). Lane 3: deglycosylated RNase

    Article Snippet: Two microliters of 500,000 units/mL PNGase F were added to 20 μ g of RNase B reconstituted in 20 μ L of commercial denaturing buffer (New England BioLabs).

    Techniques: SDS Page

    High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Journal: Nature Communications

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    doi: 10.1038/ncomms15487

    Figure Lengend Snippet: High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Article Snippet: The mixture of RNase B and fetuin was denatured by boiling for 10 min in the presence of 1 × Rapid PNGase F buffer (NEB).

    Techniques: Binding Assay, Sequencing, Incubation, SDS Page

    The Fbs1 GYR variant substantially improves N-glycopeptide enrichment. The total ion chromatogram (TIC) (upper panel) from an LC-MS analysis shows wt Fbs1 or Fbs1 GYR-mediated binding and enrichment of N-glycopeptides from a complex peptide mixture. The complex peptide mixture is a tryptic digest of RNase B spiked with SGP and SGP-TMR. RNaseB contains non-glycosylated peptides and two major high-mannose N-glycopeptides labelled in the enlargement as: M5N2-NLTK and M6N2-NLTK. M5N2 or M6N2 indicates 5 or 6 mannose residues and 2 GlcNAc residues, respectively. NLTK is the peptide sequence of the N-glycopeptide produced by trypsin treatment of RNase B. The enrichment was performed in low salt (50 mM ammonium acetate, pH7.5). The black line indicates the chromatogram of a 50% input mixture. The orange and blue lines indicate the chromatograms of Fbs1 GYR and wt Fbs1 enrichment samples. The major N-glycopeptide peaks (M5N2-NLTK, M6N2-NLTK, SGP and SGP-TMR) are indicated. N-glycopeptides were quantified from the extracted ion chromatogram of the LC-MS analysis. The ions with the correct monoisotopic m/z values, that is, M5N2-NLTK:1691.98 1- (theoretical, 1691.72 1- ), M6N2-NLTK: 1854.07 1- (theoretical,1853.72 1- ) and SGP: 1433.47 2- (theoretical, 1433.10 2- ) were extracted, integrated and quantified. The amount of SGP-TMR was determined by fluorescence measurement of the LC elution. Recovery of each N-glycopeptide (enriched peptide amount/input peptide amount) is shown as a bar graph (lower panel). A representative TIC profile is shown from three experiments.

    Journal: Nature Communications

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    doi: 10.1038/ncomms15487

    Figure Lengend Snippet: The Fbs1 GYR variant substantially improves N-glycopeptide enrichment. The total ion chromatogram (TIC) (upper panel) from an LC-MS analysis shows wt Fbs1 or Fbs1 GYR-mediated binding and enrichment of N-glycopeptides from a complex peptide mixture. The complex peptide mixture is a tryptic digest of RNase B spiked with SGP and SGP-TMR. RNaseB contains non-glycosylated peptides and two major high-mannose N-glycopeptides labelled in the enlargement as: M5N2-NLTK and M6N2-NLTK. M5N2 or M6N2 indicates 5 or 6 mannose residues and 2 GlcNAc residues, respectively. NLTK is the peptide sequence of the N-glycopeptide produced by trypsin treatment of RNase B. The enrichment was performed in low salt (50 mM ammonium acetate, pH7.5). The black line indicates the chromatogram of a 50% input mixture. The orange and blue lines indicate the chromatograms of Fbs1 GYR and wt Fbs1 enrichment samples. The major N-glycopeptide peaks (M5N2-NLTK, M6N2-NLTK, SGP and SGP-TMR) are indicated. N-glycopeptides were quantified from the extracted ion chromatogram of the LC-MS analysis. The ions with the correct monoisotopic m/z values, that is, M5N2-NLTK:1691.98 1- (theoretical, 1691.72 1- ), M6N2-NLTK: 1854.07 1- (theoretical,1853.72 1- ) and SGP: 1433.47 2- (theoretical, 1433.10 2- ) were extracted, integrated and quantified. The amount of SGP-TMR was determined by fluorescence measurement of the LC elution. Recovery of each N-glycopeptide (enriched peptide amount/input peptide amount) is shown as a bar graph (lower panel). A representative TIC profile is shown from three experiments.

    Article Snippet: The mixture of RNase B and fetuin was denatured by boiling for 10 min in the presence of 1 × Rapid PNGase F buffer (NEB).

    Techniques: Variant Assay, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Sequencing, Produced, Fluorescence

    Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.

    Journal: Nature Communications

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    doi: 10.1038/ncomms15487

    Figure Lengend Snippet: Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.

    Article Snippet: The mixture of RNase B and fetuin was denatured by boiling for 10 min in the presence of 1 × Rapid PNGase F buffer (NEB).

    Techniques: Binding Assay, Variant Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Fluorescence

    Fbs1 binds to diverse types of N-glycomolecules. ( a ) Fbs1 binding to RNase B is N-glycan dependent. RNase B or deglycosylated RNase B was subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Lane 1, RNase B input control (CTL). Lane 2, essentially no RNase B deglycosylated by PNGase F is pulled down by Fbs1. Lane 3, RNase B with N-glycans is efficiently pulled down by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 binds to the N-glycosylated heavy chain of human IgG. Denatured and reduced human IgG were subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Heavy chains of human IgGs are typically N-glycosylated. Lane 1 is a control showing the IgG light chain and heavy chain. Lane 2 is Fbs1 beads only. Some SNAP-Fbs1 protein leaches from the prototype Fbs1 beads (denoted by an asterisk). Lanes 3 and 4 show that only the glycosylated heavy chain is bound by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( c ) Fbs1 binding affinity (Kd value) to sialylglycopeptide (SGP), M3N2 and M3N2F was measured by isothermal titration calorimetry. Structures of SGP, M3N2 and M3N2F are shown in the left panel. M3N2F is M3N2 with α1-6 fucosylation at the reducing end GlcNAc. The left panel summarizes the Kd values of SGP ( n =4), M3N2 ( n =5) and M3N2F ( n =5) interacting with wt Fbs1. There is no significant difference between the Kd values of M3N2 and M3N2F ( P value 0.85 > 0.05, mean±s.e.m., t -test, two-tailed). ( d ) wt Fbs1 shows binding bias to different N-glycopeptides. SGP was labelled with TMR fluorophore to facilitate detection. N-glycans of SGP-TMR (1) were then trimmed with exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Binding of the trimmed glycopeptides to Fbs1 beads was analysed. The relative binding affinity to wt Fbs1 is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is only indicated in N-glycopeptide structure 1. Results represent the mean±s.e.m. of three replicates.

    Journal: Nature Communications

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    doi: 10.1038/ncomms15487

    Figure Lengend Snippet: Fbs1 binds to diverse types of N-glycomolecules. ( a ) Fbs1 binding to RNase B is N-glycan dependent. RNase B or deglycosylated RNase B was subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Lane 1, RNase B input control (CTL). Lane 2, essentially no RNase B deglycosylated by PNGase F is pulled down by Fbs1. Lane 3, RNase B with N-glycans is efficiently pulled down by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 binds to the N-glycosylated heavy chain of human IgG. Denatured and reduced human IgG were subjected to an Fbs1 pulldown assay and analysed by SDS–PAGE. Heavy chains of human IgGs are typically N-glycosylated. Lane 1 is a control showing the IgG light chain and heavy chain. Lane 2 is Fbs1 beads only. Some SNAP-Fbs1 protein leaches from the prototype Fbs1 beads (denoted by an asterisk). Lanes 3 and 4 show that only the glycosylated heavy chain is bound by Fbs1 beads. A representative SDS–PAGE gel is shown from two experiments. ( c ) Fbs1 binding affinity (Kd value) to sialylglycopeptide (SGP), M3N2 and M3N2F was measured by isothermal titration calorimetry. Structures of SGP, M3N2 and M3N2F are shown in the left panel. M3N2F is M3N2 with α1-6 fucosylation at the reducing end GlcNAc. The left panel summarizes the Kd values of SGP ( n =4), M3N2 ( n =5) and M3N2F ( n =5) interacting with wt Fbs1. There is no significant difference between the Kd values of M3N2 and M3N2F ( P value 0.85 > 0.05, mean±s.e.m., t -test, two-tailed). ( d ) wt Fbs1 shows binding bias to different N-glycopeptides. SGP was labelled with TMR fluorophore to facilitate detection. N-glycans of SGP-TMR (1) were then trimmed with exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Binding of the trimmed glycopeptides to Fbs1 beads was analysed. The relative binding affinity to wt Fbs1 is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is only indicated in N-glycopeptide structure 1. Results represent the mean±s.e.m. of three replicates.

    Article Snippet: The mixture of RNase B and fetuin was denatured by boiling for 10 min in the presence of 1 × Rapid PNGase F buffer (NEB).

    Techniques: Binding Assay, SDS Page, CTL Assay, Isothermal Titration Calorimetry, Two Tailed Test, Fluorescence