streptavidin agarose beads  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin agarose beads
    p62 is a target of reactive aldehydes in in vitro cell culture and human PSC. Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by <t>streptavidin</t> bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A . RAW264.7, B . HepG2. C . Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.
    Streptavidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin agarose beads/product/Thermo Fisher
    Average 97 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    streptavidin agarose beads - by Bioz Stars, 2022-11
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    Images

    1) Product Images from "The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease"

    Article Title: The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease

    Journal: PLOS ONE

    doi: 10.1371/journal.pone.0276879

    p62 is a target of reactive aldehydes in in vitro cell culture and human PSC. Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by streptavidin bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A . RAW264.7, B . HepG2. C . Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.
    Figure Legend Snippet: p62 is a target of reactive aldehydes in in vitro cell culture and human PSC. Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by streptavidin bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A . RAW264.7, B . HepG2. C . Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.

    Techniques Used: In Vitro, Cell Culture, Modification, Purification, Western Blot

    2) Product Images from "SPECC1L binds MYPT1/PP1β and can regulate its distribution between microtubules and filamentous actin"

    Article Title: SPECC1L binds MYPT1/PP1β and can regulate its distribution between microtubules and filamentous actin

    Journal: bioRxiv

    doi: 10.1101/2022.09.09.507377

    Validation of the interaction of MYPT1 with SPECC1L and SPECC1. a. Proximity biotin labeling of endogenous MYPT1 by Myc-BirA*-SPECC1L and Myc-BirA*-SPECC1 expressed in U2OS cells allows it to be captured on Streptavidin-agarose beads and visualized by WB analysis (a-b). Top panels were probed with anti-MYPT1 and bottom panels with Streptavidin-HRP. c. Only the C-terminal half of SPECC1 is able to proximity label MYPT1 when expressed as a Myc-BirA* fusion protein. The top panel was probed with anti-Myc and the bottom panel with anti-MYPT1. d. When co-expressed in cells, the GFP-MYPT1 (CD) fragment co-immunoprecipitates mCh-SPECC1L-CT. e. GFP-tagged SPECC1L and SPECC1 both co-purify endogenous MYPT1 from U2OS cell lysates. f. Only the C-terminal half of SPECC1 co-immunoprecipitates endogenous MYPT1. These data are related to Figures 1 - 2 .
    Figure Legend Snippet: Validation of the interaction of MYPT1 with SPECC1L and SPECC1. a. Proximity biotin labeling of endogenous MYPT1 by Myc-BirA*-SPECC1L and Myc-BirA*-SPECC1 expressed in U2OS cells allows it to be captured on Streptavidin-agarose beads and visualized by WB analysis (a-b). Top panels were probed with anti-MYPT1 and bottom panels with Streptavidin-HRP. c. Only the C-terminal half of SPECC1 is able to proximity label MYPT1 when expressed as a Myc-BirA* fusion protein. The top panel was probed with anti-Myc and the bottom panel with anti-MYPT1. d. When co-expressed in cells, the GFP-MYPT1 (CD) fragment co-immunoprecipitates mCh-SPECC1L-CT. e. GFP-tagged SPECC1L and SPECC1 both co-purify endogenous MYPT1 from U2OS cell lysates. f. Only the C-terminal half of SPECC1 co-immunoprecipitates endogenous MYPT1. These data are related to Figures 1 - 2 .

    Techniques Used: Labeling, Western Blot

    SPECC1L modulates the subcellular distribution and turnover dynamics of the myosin phosphatase complex. a. Mypt1-GFP (green) in the BAC HeLa line is recruited to MT bundles induced by transient overexpression of SPECC1L-BirA* (top panel, arrows), and to actin filaments at which transiently overexpressed SPECC1L-CT accumulates (lower panel, arrowheads). Biotinylation patterns were detected using AlexaFluor 647-tagged Streptavidin (red). Hoechst 33342-stained DNA is shown in blue. b. PP1β-GFP (green) in the U2OS PP1β-GFP stable line is recruited to MT bundles induced by transient overexpression of SPECC1L (top panel, arrows) and to actin stress fibers at which transiently overexpressed SPECC1L-CT accumulates (lower panel, arrowheads). Biotinylation patterns were detected using AlexaFluor 647-tagged Streptavidin (red). Hoechst 33342-stained DNA is shown in blue. c. Fluorescence Recovery After Photobleaching (FRAP) curves for GFP-tagged SPECC1L- and SPECC1L-CT (unperturbed represents SPECC1L-GFP in the U2OS SPECC1L-GFP stable line) and for Mypt1-GFP and PP1β-GFP either unperturbed or at the structures to which they are recruited by overexpressed SPECC1L or SPECC1L-CT (as shown in panels a-b). d. Tables summarizing the FRAP results, based on mobile fractions and recovery times calculated using GraphPad Prism ( Table S1 ). In all cases, the recovery curves for the mobile fraction were best fit by a double exponential line, indicating two pools with different recovery times (slow and fast). Representative FRAP experiments are shown in Fig. S4 . Scale bars are 10 μm.
    Figure Legend Snippet: SPECC1L modulates the subcellular distribution and turnover dynamics of the myosin phosphatase complex. a. Mypt1-GFP (green) in the BAC HeLa line is recruited to MT bundles induced by transient overexpression of SPECC1L-BirA* (top panel, arrows), and to actin filaments at which transiently overexpressed SPECC1L-CT accumulates (lower panel, arrowheads). Biotinylation patterns were detected using AlexaFluor 647-tagged Streptavidin (red). Hoechst 33342-stained DNA is shown in blue. b. PP1β-GFP (green) in the U2OS PP1β-GFP stable line is recruited to MT bundles induced by transient overexpression of SPECC1L (top panel, arrows) and to actin stress fibers at which transiently overexpressed SPECC1L-CT accumulates (lower panel, arrowheads). Biotinylation patterns were detected using AlexaFluor 647-tagged Streptavidin (red). Hoechst 33342-stained DNA is shown in blue. c. Fluorescence Recovery After Photobleaching (FRAP) curves for GFP-tagged SPECC1L- and SPECC1L-CT (unperturbed represents SPECC1L-GFP in the U2OS SPECC1L-GFP stable line) and for Mypt1-GFP and PP1β-GFP either unperturbed or at the structures to which they are recruited by overexpressed SPECC1L or SPECC1L-CT (as shown in panels a-b). d. Tables summarizing the FRAP results, based on mobile fractions and recovery times calculated using GraphPad Prism ( Table S1 ). In all cases, the recovery curves for the mobile fraction were best fit by a double exponential line, indicating two pools with different recovery times (slow and fast). Representative FRAP experiments are shown in Fig. S4 . Scale bars are 10 μm.

    Techniques Used: BAC Assay, Over Expression, Staining, Fluorescence

    3) Product Images from "CASP4/11 contributes to pulmonary inflammation and disease exacerbation in COVID-19"

    Article Title: CASP4/11 contributes to pulmonary inflammation and disease exacerbation in COVID-19

    Journal: medRxiv

    doi: 10.1101/2022.06.29.22277067

    Caspase-11 is important for inflammasome activation in macrophages infected with SARS-CoV-2. (A) Bone marrow derived macrophages (BMDMs) were obtained from C57BL/6, Nlrp3 −/− and Casp11 −/− mice. BMDMs were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 1 for 8h. Western blotting analysis of cell lysate was performed to evaluate caspase-11 expression after infection. (B) C57BL/6 BMDMs were primed with PAM3Cys (300 ng/mL) for 4 hours and infected with SARS-CoV-2 at a MOI of 1 for 8h. Pull-down of active caspase-11 was performed by addition of Biotin-VAD-FMK followed by Streptavidin beads. ( C ) BMDMs were obtained from C57BL/6 K18-hACE-2 and mice backcrossed with Nlrp3 −/− and Casp11 −/− mice. Cells were primed with PAM3Cys (300 ng/mL) for 4 hours and infected with SARS-CoV-2 at MOI of 1 for 24 and IL-1β levels in cell-free supernatants was measured by ELISA. (D) BMDMs from C57BL/6, Nlrp3 −/− , Casp11 −/− and Casp1/11 −/− in the K18-hACE-2 background were infected with SARS-CoV-2 at a MOI of 1 for 24h and LDH release was measured in the supernatants. * indicates P
    Figure Legend Snippet: Caspase-11 is important for inflammasome activation in macrophages infected with SARS-CoV-2. (A) Bone marrow derived macrophages (BMDMs) were obtained from C57BL/6, Nlrp3 −/− and Casp11 −/− mice. BMDMs were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 1 for 8h. Western blotting analysis of cell lysate was performed to evaluate caspase-11 expression after infection. (B) C57BL/6 BMDMs were primed with PAM3Cys (300 ng/mL) for 4 hours and infected with SARS-CoV-2 at a MOI of 1 for 8h. Pull-down of active caspase-11 was performed by addition of Biotin-VAD-FMK followed by Streptavidin beads. ( C ) BMDMs were obtained from C57BL/6 K18-hACE-2 and mice backcrossed with Nlrp3 −/− and Casp11 −/− mice. Cells were primed with PAM3Cys (300 ng/mL) for 4 hours and infected with SARS-CoV-2 at MOI of 1 for 24 and IL-1β levels in cell-free supernatants was measured by ELISA. (D) BMDMs from C57BL/6, Nlrp3 −/− , Casp11 −/− and Casp1/11 −/− in the K18-hACE-2 background were infected with SARS-CoV-2 at a MOI of 1 for 24h and LDH release was measured in the supernatants. * indicates P

    Techniques Used: Activation Assay, Infection, Derivative Assay, Mouse Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

    4) Product Images from "Pervasive translation of circular RNAs driven by short IRES-like elements"

    Article Title: Pervasive translation of circular RNAs driven by short IRES-like elements

    Journal: Nature Communications

    doi: 10.1038/s41467-022-31327-y

    Systematically identification of trans -factors that recognize IRES-like elements. Source data are provided as a Source Data file for panels ( d ) and ( e ). a Schematic diagram of RNA affinity purification. Biotin-labeled RNAs containing consensus motifs of IRES-like elements were incubated with HeLa cell lysate, and RNA-protein complexes were purified by streptavidin beads. The proteins were further identified by mass spectrometry. b Identification of trans -factors bound by each RNA probe. The probes presenting five consensus motifs of IRES-like elements (red) and a control probe (blue) were used (full sequence in Supplementary Data 2 ). The total eluded proteins were separated with SDS-PAGE, and each band was cut and analyzed by mass spectrometry. The top three identified proteins in each band were labeled. c Protein–protein interaction network of identified trans -factors. Top proteins bound by all RNA probes (i.e., IRES-like elements) were analyzed by STRING and clustered into two main groups by MCODE tool. d Validating the activity of trans -factors. The circRNA reporter inserted with two depleted hexamers in tandem were co-transfected into 293T cells with different Puf-fusion proteins that specifically recognize an 8-nt target in the inserted sequences. The resulting cells were collected at 2 days after transfection to analyze by western blot and RT-PCR. Different pairs of Puf proteins and 8-nt targets were used as specificity control. Puf-N2 specifically binds AGUGUCAG, whereas Puf-N8 specifically binds to GCGUCUGC. The bar graph represents the quantification of GFP levels relative to GAPDH. The protein levels were also normalized to the RNA (N.S. not significant). e Validation of PABPC1 activity. The expression vector of PABPC1 and various control RBPs were co-transfected with circRNA translation reporter containing (A) 10 or (G) 10 sequences before the start codon, and the protein products were assayed at 48 h after transfection. The bar graph represents the quantification of GFP levels relative to GAPDH using the western blot. The protein levels were also normalized to the RNA, and the relative amount of GFP translation was calculated by dividing the mock transfection.
    Figure Legend Snippet: Systematically identification of trans -factors that recognize IRES-like elements. Source data are provided as a Source Data file for panels ( d ) and ( e ). a Schematic diagram of RNA affinity purification. Biotin-labeled RNAs containing consensus motifs of IRES-like elements were incubated with HeLa cell lysate, and RNA-protein complexes were purified by streptavidin beads. The proteins were further identified by mass spectrometry. b Identification of trans -factors bound by each RNA probe. The probes presenting five consensus motifs of IRES-like elements (red) and a control probe (blue) were used (full sequence in Supplementary Data 2 ). The total eluded proteins were separated with SDS-PAGE, and each band was cut and analyzed by mass spectrometry. The top three identified proteins in each band were labeled. c Protein–protein interaction network of identified trans -factors. Top proteins bound by all RNA probes (i.e., IRES-like elements) were analyzed by STRING and clustered into two main groups by MCODE tool. d Validating the activity of trans -factors. The circRNA reporter inserted with two depleted hexamers in tandem were co-transfected into 293T cells with different Puf-fusion proteins that specifically recognize an 8-nt target in the inserted sequences. The resulting cells were collected at 2 days after transfection to analyze by western blot and RT-PCR. Different pairs of Puf proteins and 8-nt targets were used as specificity control. Puf-N2 specifically binds AGUGUCAG, whereas Puf-N8 specifically binds to GCGUCUGC. The bar graph represents the quantification of GFP levels relative to GAPDH. The protein levels were also normalized to the RNA (N.S. not significant). e Validation of PABPC1 activity. The expression vector of PABPC1 and various control RBPs were co-transfected with circRNA translation reporter containing (A) 10 or (G) 10 sequences before the start codon, and the protein products were assayed at 48 h after transfection. The bar graph represents the quantification of GFP levels relative to GAPDH using the western blot. The protein levels were also normalized to the RNA, and the relative amount of GFP translation was calculated by dividing the mock transfection.

    Techniques Used: Affinity Purification, Labeling, Incubation, Purification, Mass Spectrometry, Sequencing, SDS Page, Activity Assay, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation

    5) Product Images from "Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation"

    Article Title: Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation

    Journal: eLife

    doi: 10.7554/eLife.79990

    Virus internalization by langerin. ( A ) Surface labeling of langerin. Langerin-expressing cells were surface-labeled using biotin N-hydroxysulfosuccinimide ester, and labeled langerin were immunoprecipitated using streptavidin agarose. ‘Int’ and ‘Sur’ indicate intracellular and surface langerin, respectively. The intracellular: surface ratio of langerin was approximately 5:1 and this ratio was not significantly affected by mutations. ( B ) Quantification of unbound viruses using p24 ELISA. Medium supernatants were diluted 1000-fold before loading into the ELISA plate. Concentration of viruses in the medium supernatant were nearly equal among the wild type and the mutants. ( C ) Anti-langerin immunoblot of stably-expressing cell line. The expression level of langerin in the stable cell line was approximately 30% of that of transiently-expressing cells. Given that the transformation efficiency of transiently-expressing cells was 40%, the actual expression level of langerin per cell is estimated to be ~12%. ( D ) Electron microscopy of the stable cell line. Birbeck granule formation was induced by addition of yeast mannan. Short and isolated Birbeck granules were observed (arrow) ( E ) Quantification of Birbeck granule formation. ‘Transient’ corresponds to WT mannan (+) in Figure 4B–D . 74 Birbeck granules in 20 stably-expressing cells were measured. p=2.0 × 10 –30 (individual length), 2.5×10 –12 (individual length), and 9.7×10 –14 (number). Original blot image of Figure 5—figure supplement 1A . Annotated blot image of Figure 5—figure supplement 1A . Original blot image of Figure 5—figure supplement 1C (anti-langerin). Original blot image of Figure 5—figure supplement 1C (anti-tubulin).
    Figure Legend Snippet: Virus internalization by langerin. ( A ) Surface labeling of langerin. Langerin-expressing cells were surface-labeled using biotin N-hydroxysulfosuccinimide ester, and labeled langerin were immunoprecipitated using streptavidin agarose. ‘Int’ and ‘Sur’ indicate intracellular and surface langerin, respectively. The intracellular: surface ratio of langerin was approximately 5:1 and this ratio was not significantly affected by mutations. ( B ) Quantification of unbound viruses using p24 ELISA. Medium supernatants were diluted 1000-fold before loading into the ELISA plate. Concentration of viruses in the medium supernatant were nearly equal among the wild type and the mutants. ( C ) Anti-langerin immunoblot of stably-expressing cell line. The expression level of langerin in the stable cell line was approximately 30% of that of transiently-expressing cells. Given that the transformation efficiency of transiently-expressing cells was 40%, the actual expression level of langerin per cell is estimated to be ~12%. ( D ) Electron microscopy of the stable cell line. Birbeck granule formation was induced by addition of yeast mannan. Short and isolated Birbeck granules were observed (arrow) ( E ) Quantification of Birbeck granule formation. ‘Transient’ corresponds to WT mannan (+) in Figure 4B–D . 74 Birbeck granules in 20 stably-expressing cells were measured. p=2.0 × 10 –30 (individual length), 2.5×10 –12 (individual length), and 9.7×10 –14 (number). Original blot image of Figure 5—figure supplement 1A . Annotated blot image of Figure 5—figure supplement 1A . Original blot image of Figure 5—figure supplement 1C (anti-langerin). Original blot image of Figure 5—figure supplement 1C (anti-tubulin).

    Techniques Used: Labeling, Expressing, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Stable Transfection, Transformation Assay, Electron Microscopy, Isolation

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    Thermo Fisher affinity streptavidin agarose beads
    Affinity Streptavidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/affinity streptavidin agarose beads/product/Thermo Fisher
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    Thermo Fisher high capacity streptavidin agarose beads
    Design of the synthetic combinatorial peptide library and workflow to determine the activity and prime-side specificity of a Pro-Pro endopeptidase (PPEP). The library was designed to contain an XXPPXX motif, X representing any residue (X≠Cys). First, the library was pre-cleaned on avidin <t>beads</t> to remove non-biotinylated peptides. Then, the library was incubated with a PPEP. The scissile bond is indicated by the arrow. Following this, biotinylated peptides (non-cleaved peptides and N-terminal product peptides) were captured on a <t>streptavidin</t> column. The flow-through, containing non-biotinylated C-terminal product peptides (PXXGGLEEF) were then analyzed by LC-MS/MS, after which the primeside specificity could be determined. Ahx: 1-aminohexanoic acid.
    High Capacity Streptavidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity streptavidin agarose beads/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    88
    Thermo Fisher streptavidin agarose beads
    p62 is a target of reactive aldehydes in in vitro cell culture and human PSC. Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by <t>streptavidin</t> bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A . RAW264.7, B . HepG2. C . Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.
    Streptavidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin agarose beads/product/Thermo Fisher
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin agarose beads - by Bioz Stars, 2022-11
    88/100 stars
      Buy from Supplier

    88
    Thermo Fisher monomeric avidin agarose beads
    p62 is a target of reactive aldehydes in in vitro cell culture and human PSC. Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by <t>streptavidin</t> bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A . RAW264.7, B . HepG2. C . Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.
    Monomeric Avidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monomeric avidin agarose beads/product/Thermo Fisher
    Average 88 stars, based on 1 article reviews
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    Design of the synthetic combinatorial peptide library and workflow to determine the activity and prime-side specificity of a Pro-Pro endopeptidase (PPEP). The library was designed to contain an XXPPXX motif, X representing any residue (X≠Cys). First, the library was pre-cleaned on avidin beads to remove non-biotinylated peptides. Then, the library was incubated with a PPEP. The scissile bond is indicated by the arrow. Following this, biotinylated peptides (non-cleaved peptides and N-terminal product peptides) were captured on a streptavidin column. The flow-through, containing non-biotinylated C-terminal product peptides (PXXGGLEEF) were then analyzed by LC-MS/MS, after which the primeside specificity could be determined. Ahx: 1-aminohexanoic acid.

    Journal: bioRxiv

    Article Title: In-depth specificity profiling of Pro-Pro endopeptidases (PPEPs) using combinatorial synthetic peptide libraries

    doi: 10.1101/2022.11.15.516584

    Figure Lengend Snippet: Design of the synthetic combinatorial peptide library and workflow to determine the activity and prime-side specificity of a Pro-Pro endopeptidase (PPEP). The library was designed to contain an XXPPXX motif, X representing any residue (X≠Cys). First, the library was pre-cleaned on avidin beads to remove non-biotinylated peptides. Then, the library was incubated with a PPEP. The scissile bond is indicated by the arrow. Following this, biotinylated peptides (non-cleaved peptides and N-terminal product peptides) were captured on a streptavidin column. The flow-through, containing non-biotinylated C-terminal product peptides (PXXGGLEEF) were then analyzed by LC-MS/MS, after which the primeside specificity could be determined. Ahx: 1-aminohexanoic acid.

    Article Snippet: After incubation, the samples were loaded onto an in-house constructed column consisting of a 200 μl pipette tip containing a filter and a packed column of 100 μl of Pierce High Capacity Streptavidin Agarose beads (Thermo, column was washed four times with 150 μl PBS prior to use), in order to remove the biotinylated peptides.

    Techniques: Activity Assay, Avidin-Biotin Assay, Incubation, Liquid Chromatography with Mass Spectroscopy

    p62 is a target of reactive aldehydes in in vitro cell culture and human PSC. Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by streptavidin bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A . RAW264.7, B . HepG2. C . Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.

    Journal: PLOS ONE

    Article Title: The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease

    doi: 10.1371/journal.pone.0276879

    Figure Lengend Snippet: p62 is a target of reactive aldehydes in in vitro cell culture and human PSC. Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by streptavidin bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A . RAW264.7, B . HepG2. C . Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.

    Article Snippet: Biotinylated proteins were purified by incubating with streptavidin agarose beads (Thermo Fisher) on a rotary mixer at 4°C O/N.

    Techniques: In Vitro, Cell Culture, Modification, Purification, Western Blot