molecular affinity fingerprint Search Results


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  • 99
    ATCC bacillus cereus atcc 14579
    Bacillus Cereus Atcc 14579, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 741 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin agarose beads
    Functional analysis of Zn 2+ -finger and MPN domain mutations a , Unfolding of poly-ubiquitinated Eos by wild-type S. cerevisiae . b, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated Zn 2+ -finger mutants, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37°C) or three days (25°C). c , As in a . d , Binding of poly-ubiquitinated substrate to SBP-tagged C. thermophilum Npl4 (Zn 2+ -finger/MPN/CTD domains, residues 129-602) or the indicated variants (MPN only: residues 129-519, CTD only: residues 519-602). The bait proteins were bound to <t>streptavidin</t> beads and incubated with dye-labeled, poly-ubiquitinated superfolder GFP. Bound material was analyzed by SDS-PAGE followed by fluorescence scanning (top) and Coomassie blue staining (bottom). M, molecular weight markers. e , The locations of MPN cleft mutants tested in d are shown in stick representation. The MPN, insert-1, insert-2, and CTD are shown in tan, magenta, purple, and orange, respectively.
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    Thermo Fisher streptavidin plus ultralink resin
    Functional analysis of Zn 2+ -finger and MPN domain mutations a , Unfolding of poly-ubiquitinated Eos by wild-type S. cerevisiae . b, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated Zn 2+ -finger mutants, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37°C) or three days (25°C). c , As in a . d , Binding of poly-ubiquitinated substrate to SBP-tagged C. thermophilum Npl4 (Zn 2+ -finger/MPN/CTD domains, residues 129-602) or the indicated variants (MPN only: residues 129-519, CTD only: residues 519-602). The bait proteins were bound to <t>streptavidin</t> beads and incubated with dye-labeled, poly-ubiquitinated superfolder GFP. Bound material was analyzed by SDS-PAGE followed by fluorescence scanning (top) and Coomassie blue staining (bottom). M, molecular weight markers. e , The locations of MPN cleft mutants tested in d are shown in stick representation. The MPN, insert-1, insert-2, and CTD are shown in tan, magenta, purple, and orange, respectively.
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    Thermo Fisher egf
    Functional analysis of Zn 2+ -finger and MPN domain mutations a , Unfolding of poly-ubiquitinated Eos by wild-type S. cerevisiae . b, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated Zn 2+ -finger mutants, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37°C) or three days (25°C). c , As in a . d , Binding of poly-ubiquitinated substrate to SBP-tagged C. thermophilum Npl4 (Zn 2+ -finger/MPN/CTD domains, residues 129-602) or the indicated variants (MPN only: residues 129-519, CTD only: residues 519-602). The bait proteins were bound to <t>streptavidin</t> beads and incubated with dye-labeled, poly-ubiquitinated superfolder GFP. Bound material was analyzed by SDS-PAGE followed by fluorescence scanning (top) and Coomassie blue staining (bottom). M, molecular weight markers. e , The locations of MPN cleft mutants tested in d are shown in stick representation. The MPN, insert-1, insert-2, and CTD are shown in tan, magenta, purple, and orange, respectively.
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    Promega magnetic streptavidin coated beads
    Functional analysis of Zn 2+ -finger and MPN domain mutations a , Unfolding of poly-ubiquitinated Eos by wild-type S. cerevisiae . b, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated Zn 2+ -finger mutants, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37°C) or three days (25°C). c , As in a . d , Binding of poly-ubiquitinated substrate to SBP-tagged C. thermophilum Npl4 (Zn 2+ -finger/MPN/CTD domains, residues 129-602) or the indicated variants (MPN only: residues 129-519, CTD only: residues 519-602). The bait proteins were bound to <t>streptavidin</t> beads and incubated with dye-labeled, poly-ubiquitinated superfolder GFP. Bound material was analyzed by SDS-PAGE followed by fluorescence scanning (top) and Coomassie blue staining (bottom). M, molecular weight markers. e , The locations of MPN cleft mutants tested in d are shown in stick representation. The MPN, insert-1, insert-2, and CTD are shown in tan, magenta, purple, and orange, respectively.
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    Millipore trypsin
    Functional analysis of Zn 2+ -finger and MPN domain mutations a , Unfolding of poly-ubiquitinated Eos by wild-type S. cerevisiae . b, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated Zn 2+ -finger mutants, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37°C) or three days (25°C). c , As in a . d , Binding of poly-ubiquitinated substrate to SBP-tagged C. thermophilum Npl4 (Zn 2+ -finger/MPN/CTD domains, residues 129-602) or the indicated variants (MPN only: residues 129-519, CTD only: residues 519-602). The bait proteins were bound to <t>streptavidin</t> beads and incubated with dye-labeled, poly-ubiquitinated superfolder GFP. Bound material was analyzed by SDS-PAGE followed by fluorescence scanning (top) and Coomassie blue staining (bottom). M, molecular weight markers. e , The locations of MPN cleft mutants tested in d are shown in stick representation. The MPN, insert-1, insert-2, and CTD are shown in tan, magenta, purple, and orange, respectively.
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    Thermo Fisher high capacity streptavidin agarose
    BAX Interaction Site Analysis by PUMA SAHB Photoaffinity Labeling and Mass Spectrometry (A) PUMA pSAHB-1 was incubated with full-length BAX and the mixture subjected to UV irradiation, <t>streptavidin</t> pulldown, electrophoresis, excision of the crosslinked protein, trypsin proteolysis, and LC-MS/MS analysis. A single arginine substitution (A150R) was made in PUMA pSAHB-1 to facilitate tryptic digestion into shorter and more identifiable fragments by MS. The plot (left) depicts the frequency of crosslinked sites identified across the BAX polypeptide sequence, with crosslinked residues mapped onto the solution structure of monomeric BAX (PDB ID 2K7W) (right) and colored according to the frequency of occurrence. The C-terminal α9 helix of BAX has been removed from the structure to better visualize the crosslinked residues of the canonical BH3-binding pocket. (B–D) The corresponding analysis was performed for PUMA pSAHB-2 with full length BAX (B), PUMA pSAHB-1 with C-terminal helix-deleted BAX (BAXΔC) (C), and PUMA pSAHB-2 with BAXΔC (D). X, stapling amino acid; B, norleucine; U, 4-benzoyl-phenylalanine (Bpa). .
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    Jackson Immuno alkaline phosphatase anti mouse igg
    BAX Interaction Site Analysis by PUMA SAHB Photoaffinity Labeling and Mass Spectrometry (A) PUMA pSAHB-1 was incubated with full-length BAX and the mixture subjected to UV irradiation, <t>streptavidin</t> pulldown, electrophoresis, excision of the crosslinked protein, trypsin proteolysis, and LC-MS/MS analysis. A single arginine substitution (A150R) was made in PUMA pSAHB-1 to facilitate tryptic digestion into shorter and more identifiable fragments by MS. The plot (left) depicts the frequency of crosslinked sites identified across the BAX polypeptide sequence, with crosslinked residues mapped onto the solution structure of monomeric BAX (PDB ID 2K7W) (right) and colored according to the frequency of occurrence. The C-terminal α9 helix of BAX has been removed from the structure to better visualize the crosslinked residues of the canonical BH3-binding pocket. (B–D) The corresponding analysis was performed for PUMA pSAHB-2 with full length BAX (B), PUMA pSAHB-1 with C-terminal helix-deleted BAX (BAXΔC) (C), and PUMA pSAHB-2 with BAXΔC (D). X, stapling amino acid; B, norleucine; U, 4-benzoyl-phenylalanine (Bpa). .
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    JASCO Inc far uv circular dichroism
    BAX Interaction Site Analysis by PUMA SAHB Photoaffinity Labeling and Mass Spectrometry (A) PUMA pSAHB-1 was incubated with full-length BAX and the mixture subjected to UV irradiation, <t>streptavidin</t> pulldown, electrophoresis, excision of the crosslinked protein, trypsin proteolysis, and LC-MS/MS analysis. A single arginine substitution (A150R) was made in PUMA pSAHB-1 to facilitate tryptic digestion into shorter and more identifiable fragments by MS. The plot (left) depicts the frequency of crosslinked sites identified across the BAX polypeptide sequence, with crosslinked residues mapped onto the solution structure of monomeric BAX (PDB ID 2K7W) (right) and colored according to the frequency of occurrence. The C-terminal α9 helix of BAX has been removed from the structure to better visualize the crosslinked residues of the canonical BH3-binding pocket. (B–D) The corresponding analysis was performed for PUMA pSAHB-2 with full length BAX (B), PUMA pSAHB-1 with C-terminal helix-deleted BAX (BAXΔC) (C), and PUMA pSAHB-2 with BAXΔC (D). X, stapling amino acid; B, norleucine; U, 4-benzoyl-phenylalanine (Bpa). .
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    Thermo Fisher probond nickel chelating resin
    BAX Interaction Site Analysis by PUMA SAHB Photoaffinity Labeling and Mass Spectrometry (A) PUMA pSAHB-1 was incubated with full-length BAX and the mixture subjected to UV irradiation, <t>streptavidin</t> pulldown, electrophoresis, excision of the crosslinked protein, trypsin proteolysis, and LC-MS/MS analysis. A single arginine substitution (A150R) was made in PUMA pSAHB-1 to facilitate tryptic digestion into shorter and more identifiable fragments by MS. The plot (left) depicts the frequency of crosslinked sites identified across the BAX polypeptide sequence, with crosslinked residues mapped onto the solution structure of monomeric BAX (PDB ID 2K7W) (right) and colored according to the frequency of occurrence. The C-terminal α9 helix of BAX has been removed from the structure to better visualize the crosslinked residues of the canonical BH3-binding pocket. (B–D) The corresponding analysis was performed for PUMA pSAHB-2 with full length BAX (B), PUMA pSAHB-1 with C-terminal helix-deleted BAX (BAXΔC) (C), and PUMA pSAHB-2 with BAXΔC (D). X, stapling amino acid; B, norleucine; U, 4-benzoyl-phenylalanine (Bpa). .
    Probond Nickel Chelating Resin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 752 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare heparin column
    BAX Interaction Site Analysis by PUMA SAHB Photoaffinity Labeling and Mass Spectrometry (A) PUMA pSAHB-1 was incubated with full-length BAX and the mixture subjected to UV irradiation, <t>streptavidin</t> pulldown, electrophoresis, excision of the crosslinked protein, trypsin proteolysis, and LC-MS/MS analysis. A single arginine substitution (A150R) was made in PUMA pSAHB-1 to facilitate tryptic digestion into shorter and more identifiable fragments by MS. The plot (left) depicts the frequency of crosslinked sites identified across the BAX polypeptide sequence, with crosslinked residues mapped onto the solution structure of monomeric BAX (PDB ID 2K7W) (right) and colored according to the frequency of occurrence. The C-terminal α9 helix of BAX has been removed from the structure to better visualize the crosslinked residues of the canonical BH3-binding pocket. (B–D) The corresponding analysis was performed for PUMA pSAHB-2 with full length BAX (B), PUMA pSAHB-1 with C-terminal helix-deleted BAX (BAXΔC) (C), and PUMA pSAHB-2 with BAXΔC (D). X, stapling amino acid; B, norleucine; U, 4-benzoyl-phenylalanine (Bpa). .
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    Epitomics rabbit monoclonal anti prdx6 antibody
    Discovery of peroxiredoxin 6 <t>(PRDX6)</t> as a candidate biomarker for brain injury. Rat brain proteome was fractionated by two-dimensional gel electrophoresis and transferred to polyvinylidene difluoride (PVDF). Blots were probed with serum from control and traumatic brain injury (TBI) rats (1:250), and visualized by enhanced chemiluminescence using pooled anti-rat immunoglobulin G (IgG) and IgM detection antibodies ( A and B , respectively). A feature showing enhanced autoreactivity following TBI (circles) was mapped to a replicate protein gel (C) and identified by peptide mass fingerprinting as PRDX6 (D) . (E) A representative standard curve for the sandwich immunosorbent electrochemiluminescence assay (IEA) developed to measure PRDX6 in human samples. The data reflect findings involving the independent two-dimensional gel analysis of six different pools of control and TBI serum performed in duplicate. Autoimmune signals were mapped to PRDX6 in the duplicate analyses of three of the six pools.
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    Millipore agarose gel
    Discovery of peroxiredoxin 6 <t>(PRDX6)</t> as a candidate biomarker for brain injury. Rat brain proteome was fractionated by two-dimensional gel electrophoresis and transferred to polyvinylidene difluoride (PVDF). Blots were probed with serum from control and traumatic brain injury (TBI) rats (1:250), and visualized by enhanced chemiluminescence using pooled anti-rat immunoglobulin G (IgG) and IgM detection antibodies ( A and B , respectively). A feature showing enhanced autoreactivity following TBI (circles) was mapped to a replicate protein gel (C) and identified by peptide mass fingerprinting as PRDX6 (D) . (E) A representative standard curve for the sandwich immunosorbent electrochemiluminescence assay (IEA) developed to measure PRDX6 in human samples. The data reflect findings involving the independent two-dimensional gel analysis of six different pools of control and TBI serum performed in duplicate. Autoimmune signals were mapped to PRDX6 in the duplicate analyses of three of the six pools.
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    Millipore agarose
    Discovery of peroxiredoxin 6 <t>(PRDX6)</t> as a candidate biomarker for brain injury. Rat brain proteome was fractionated by two-dimensional gel electrophoresis and transferred to polyvinylidene difluoride (PVDF). Blots were probed with serum from control and traumatic brain injury (TBI) rats (1:250), and visualized by enhanced chemiluminescence using pooled anti-rat immunoglobulin G (IgG) and IgM detection antibodies ( A and B , respectively). A feature showing enhanced autoreactivity following TBI (circles) was mapped to a replicate protein gel (C) and identified by peptide mass fingerprinting as PRDX6 (D) . (E) A representative standard curve for the sandwich immunosorbent electrochemiluminescence assay (IEA) developed to measure PRDX6 in human samples. The data reflect findings involving the independent two-dimensional gel analysis of six different pools of control and TBI serum performed in duplicate. Autoimmune signals were mapped to PRDX6 in the duplicate analyses of three of the six pools.
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    Glycoform e selectin ligand
    Expression of the ∼65-kDa <t>E-selectin</t> ligand is characteristic of MLs and circulating myeloid cells during leukemoid reactions (FL), and comprises the heavy chain of MPO. ( A ) Lysates of mobilized leukocytes (MLs), native leukocytes (NLs), and circulating myeloid cells of patients with febrile leukocytosis (FL), normalized for input cell number, were resolved by SDS/PAGE and stained in Western blot with E-selectin Ig chimera (E–Ig). A representative blot of multiple patient samples ( n > 14 for each group) is shown. Expression of the ∼65-kDa glycoprotein is characteristic of G-CSF–primed cells (MLs and FLs), but not of NLs. ( B ) Lysates of MLs (lane 1) and WGA lectin chromatography-purified glycoproteins (lane 2) resolved on reducing 7.5% SDS/PAGE gel and immunoblotted with E-selectin–Ig chimera. E-selectin ligands are present at ∼140 kDa (PSGL-1), ∼100 kDa (HCELL), and at ∼65 kDa. ( C ) Representative results of Western blots of MPO immunoprecipitates (IPs) from MLs resolved under nonreducing (lane 1) or reducing conditions (lanes 2 and 3) and stained with anti-MPO mAb. In nonreduced gels, the mature homodimer of ∼140 kDa is evident (lane 1). Under reducing conditions, Western blot with anti-MPO mAb 2C7 (lane 2) reveals bands at ∼90 kDa (precursor) and ∼65 kDa (heavy chain) or only the ∼65-kDa band when stained with anti-MPO mAb 3D3 (lane 3).
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    GenWay mouse anti prdx6 monoclonal antibody
    Discovery of peroxiredoxin 6 <t>(PRDX6)</t> as a candidate biomarker for brain injury. Rat brain proteome was fractionated by two-dimensional gel electrophoresis and transferred to polyvinylidene difluoride (PVDF). Blots were probed with serum from control and traumatic brain injury (TBI) rats (1:250), and visualized by enhanced chemiluminescence using pooled anti-rat immunoglobulin G (IgG) and IgM detection antibodies ( A and B , respectively). A feature showing enhanced autoreactivity following TBI (circles) was mapped to a replicate protein gel (C) and identified by peptide mass fingerprinting as PRDX6 (D) . (E) A representative standard curve for the sandwich immunosorbent electrochemiluminescence assay (IEA) developed to measure PRDX6 in human samples. The data reflect findings involving the independent two-dimensional gel analysis of six different pools of control and TBI serum performed in duplicate. Autoimmune signals were mapped to PRDX6 in the duplicate analyses of three of the six pools.
    Mouse Anti Prdx6 Monoclonal Antibody, supplied by GenWay, used in various techniques. Bioz Stars score: 90/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    US Biological Life Sciences mouse monoclonal anti pap antibody
    Identification of proteins in fraction III. A. MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. B. <t>Immunoblot</t> analysis of fraction III probed with <t>anti-PAP</t> antibody. Molecular weight markers shown are in kDa.
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    iNovacia hfe
    Identification of proteins in fraction III. A. MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. B. <t>Immunoblot</t> analysis of fraction III probed with <t>anti-PAP</t> antibody. Molecular weight markers shown are in kDa.
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    Thermo Fisher nacl
    Identification of proteins in fraction III. A. MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. B. <t>Immunoblot</t> analysis of fraction III probed with <t>anti-PAP</t> antibody. Molecular weight markers shown are in kDa.
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    GE Healthcare ni affinity chromatography
    Identification of proteins in fraction III. A. MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. B. <t>Immunoblot</t> analysis of fraction III probed with <t>anti-PAP</t> antibody. Molecular weight markers shown are in kDa.
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    Image Search Results


    Functional analysis of Zn 2+ -finger and MPN domain mutations a , Unfolding of poly-ubiquitinated Eos by wild-type S. cerevisiae . b, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated Zn 2+ -finger mutants, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37°C) or three days (25°C). c , As in a . d , Binding of poly-ubiquitinated substrate to SBP-tagged C. thermophilum Npl4 (Zn 2+ -finger/MPN/CTD domains, residues 129-602) or the indicated variants (MPN only: residues 129-519, CTD only: residues 519-602). The bait proteins were bound to streptavidin beads and incubated with dye-labeled, poly-ubiquitinated superfolder GFP. Bound material was analyzed by SDS-PAGE followed by fluorescence scanning (top) and Coomassie blue staining (bottom). M, molecular weight markers. e , The locations of MPN cleft mutants tested in d are shown in stick representation. The MPN, insert-1, insert-2, and CTD are shown in tan, magenta, purple, and orange, respectively.

    Journal: Nature structural & molecular biology

    Article Title: Structure of the Cdc48 ATPase with its ubiquitin-binding cofactor Ufd1-Npl4

    doi: 10.1038/s41594-018-0085-x

    Figure Lengend Snippet: Functional analysis of Zn 2+ -finger and MPN domain mutations a , Unfolding of poly-ubiquitinated Eos by wild-type S. cerevisiae . b, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated Zn 2+ -finger mutants, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37°C) or three days (25°C). c , As in a . d , Binding of poly-ubiquitinated substrate to SBP-tagged C. thermophilum Npl4 (Zn 2+ -finger/MPN/CTD domains, residues 129-602) or the indicated variants (MPN only: residues 129-519, CTD only: residues 519-602). The bait proteins were bound to streptavidin beads and incubated with dye-labeled, poly-ubiquitinated superfolder GFP. Bound material was analyzed by SDS-PAGE followed by fluorescence scanning (top) and Coomassie blue staining (bottom). M, molecular weight markers. e , The locations of MPN cleft mutants tested in d are shown in stick representation. The MPN, insert-1, insert-2, and CTD are shown in tan, magenta, purple, and orange, respectively.

    Article Snippet: The C. thermophilum Npl4 Zn2+ -finger/MPN/CTD fragments (residues 129-602, 129-519, or 519-602) with an N-terminal SBP tag were incubated with streptavidin agarose beads (Pierce) in binding buffer (50 mM Tris pH 8.0, 150 mM NaCl) for 30 min at room temperature.

    Techniques: Functional Assay, Transformation Assay, Plasmid Preparation, Serial Dilution, Incubation, Binding Assay, Labeling, SDS Page, Fluorescence, Staining, Molecular Weight

    Time-dependent association of antigen-B cell receptor complexes with MHC class II molecules. A , diagram of the approach. B cells are pulsed with biotin-labeled BCR ligand (antigen or anti-BCR mAb). Cells are lysed, and biotin-labeled BCR ligand (and associated proteins) are recovered by pulldown with streptavidin beads. Pulldowns are probed for MHC class II by Western blot analysis for class II β chain cytoplasmic tail (anti-class II). B , A20μWT B cells were pulsed with PC-BSA-btn for the indicated times and then analyzed for PC-BSA-btn—BCR—class II complexes ( cmplx ) as diagrammed in A . WCL was also analyzed for class II. Shown are representative results from one of six independent experiments. Isolated complexes and WCL were also probed for transferrin receptor and GAPDH. Shown are representative results from one of three independent experiments. In all experiments, both cmplx and WCL blots for each marker ( e.g. class II) were developed with the same ECL reagent and exposed for the same time. C , quantitative analysis of average level of PC-BSA-btn—BCR—class II complexes across six independent experiments. D and E , splenic B cells were pulsed with anti-IgM-btn and ligand-BCR-class II complexes detected as in B and C (quantitation of results from three independent experiments). Isolated complexes and WCL were also probed for the IgM heavy chain of the BCR ( BCR Blots ). The difference in signal between “No Ligand” and T = 0 in the WCL is likely due to ligation-induced association of some BCR molecules with detergent-insoluble cellular elements such as the cytoskeleton. F , 1D6 A10 CD79A-YFP cells were pulsed with anti-IgM-btn for the indicated time, lysed in Brig-58 lysis buffer, and BCR—anti-IgM-btn was pulled down with streptavidin beads. The pulldown ( Cmplx ) and WCL were then analyzed by Western blotting with anti-YFP antibody. Shown are representative results from one of two independent experiments. G , A20μWT B cells were cultured in the continued presence of low levels of biotin-labeled F(ab') 2 fragments of goat anti-human IgM antibody for the indicated times. The cells were analyzed for ligand-BCR-class II complexes as in B . Parallel streptavidin pulldown samples were also analyzed for anti-BCR-btn by streptavidin-HRP blotting. Closed and open arrowheads indicate the position of the anti-BCR-btn antibody (heavy chain F(ab') 2 fragment and/or intact light chain) and proteolytic breakdown fragments, respectively. The position of the molecular weight markers (in kilodaltons) is shown. The increase in signal for the intact heavy chain and CD79-YFP ( F ) from 0–30 min is due to ligand binding to intracellular receptors that are recruited to the cell surface upon exposure to BCR ligand (data not shown). Shown are representative results from one of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules *

    doi: 10.1074/jbc.M115.649582

    Figure Lengend Snippet: Time-dependent association of antigen-B cell receptor complexes with MHC class II molecules. A , diagram of the approach. B cells are pulsed with biotin-labeled BCR ligand (antigen or anti-BCR mAb). Cells are lysed, and biotin-labeled BCR ligand (and associated proteins) are recovered by pulldown with streptavidin beads. Pulldowns are probed for MHC class II by Western blot analysis for class II β chain cytoplasmic tail (anti-class II). B , A20μWT B cells were pulsed with PC-BSA-btn for the indicated times and then analyzed for PC-BSA-btn—BCR—class II complexes ( cmplx ) as diagrammed in A . WCL was also analyzed for class II. Shown are representative results from one of six independent experiments. Isolated complexes and WCL were also probed for transferrin receptor and GAPDH. Shown are representative results from one of three independent experiments. In all experiments, both cmplx and WCL blots for each marker ( e.g. class II) were developed with the same ECL reagent and exposed for the same time. C , quantitative analysis of average level of PC-BSA-btn—BCR—class II complexes across six independent experiments. D and E , splenic B cells were pulsed with anti-IgM-btn and ligand-BCR-class II complexes detected as in B and C (quantitation of results from three independent experiments). Isolated complexes and WCL were also probed for the IgM heavy chain of the BCR ( BCR Blots ). The difference in signal between “No Ligand” and T = 0 in the WCL is likely due to ligation-induced association of some BCR molecules with detergent-insoluble cellular elements such as the cytoskeleton. F , 1D6 A10 CD79A-YFP cells were pulsed with anti-IgM-btn for the indicated time, lysed in Brig-58 lysis buffer, and BCR—anti-IgM-btn was pulled down with streptavidin beads. The pulldown ( Cmplx ) and WCL were then analyzed by Western blotting with anti-YFP antibody. Shown are representative results from one of two independent experiments. G , A20μWT B cells were cultured in the continued presence of low levels of biotin-labeled F(ab') 2 fragments of goat anti-human IgM antibody for the indicated times. The cells were analyzed for ligand-BCR-class II complexes as in B . Parallel streptavidin pulldown samples were also analyzed for anti-BCR-btn by streptavidin-HRP blotting. Closed and open arrowheads indicate the position of the anti-BCR-btn antibody (heavy chain F(ab') 2 fragment and/or intact light chain) and proteolytic breakdown fragments, respectively. The position of the molecular weight markers (in kilodaltons) is shown. The increase in signal for the intact heavy chain and CD79-YFP ( F ) from 0–30 min is due to ligand binding to intracellular receptors that are recruited to the cell surface upon exposure to BCR ligand (data not shown). Shown are representative results from one of three independent experiments.

    Article Snippet: Btn-ligand-BCR—class II complexes were recovered by pulldown with 100 μl of a 10% suspension of streptavidin-agarose (Thermo Scientific, catalog no. 20353) overnight at 4 °C with constant inversion.

    Techniques: Labeling, Western Blot, Isolation, Marker, Quantitation Assay, Ligation, Lysis, Cell Culture, Molecular Weight, Ligand Binding Assay

    Binding of carboxy-terminal L2 peptides to retromer. A . The top lines show the sequences of biotinylated peptides, where B indicates position of biotin. Putative retromer recognition motifs in the carboxy-terminal peptide are shown in boxes. Mutant versions of the carboxy-terminal peptide are also shown, with the mutations in red. B . Left panel. L2-N, L2-M, or L2-C peptide was incubated with uninfected HeLa cell RIPA lysate. The samples were analyzed by streptavidin pull-down, SDS-PAGE, and immunoblotting with an anti-Vps35 antibody. Molecular weight markers in kDa are shown at the left. Right panel. The wild-type or a mutant carboxy-terminal peptide were incubated with uninfected HeLa cell HEPES lysate and processed as in panel A. Similar results were obtained in three or more independent experiments. C . Experiments performed as in panel B with HaCaT cell RIPA lysates.

    Journal: PLoS Pathogens

    Article Title: Direct Binding of Retromer to Human Papillomavirus Type 16 Minor Capsid Protein L2 Mediates Endosome Exit during Viral Infection

    doi: 10.1371/journal.ppat.1004699

    Figure Lengend Snippet: Binding of carboxy-terminal L2 peptides to retromer. A . The top lines show the sequences of biotinylated peptides, where B indicates position of biotin. Putative retromer recognition motifs in the carboxy-terminal peptide are shown in boxes. Mutant versions of the carboxy-terminal peptide are also shown, with the mutations in red. B . Left panel. L2-N, L2-M, or L2-C peptide was incubated with uninfected HeLa cell RIPA lysate. The samples were analyzed by streptavidin pull-down, SDS-PAGE, and immunoblotting with an anti-Vps35 antibody. Molecular weight markers in kDa are shown at the left. Right panel. The wild-type or a mutant carboxy-terminal peptide were incubated with uninfected HeLa cell HEPES lysate and processed as in panel A. Similar results were obtained in three or more independent experiments. C . Experiments performed as in panel B with HaCaT cell RIPA lysates.

    Article Snippet: 40 μl of streptavidin agarose beads slurry (Pierce, cat# 20349) was added, and the mixture was gently rocked for 45 min at 4°C.

    Techniques: Binding Assay, Mutagenesis, Incubation, SDS Page, Molecular Weight

    Effect of GRC antisense expression on BIO14.6 myotubes. (a) Immunoblot assay for GRC and β-dystroglycan (β-DG; top) and GRC immunohistochemistry (middle) of BIO14.6 myotubes infected with Ad.β-gal or Ad-antisense–GRC cDNA (Ad.asGRC). Cell surface GRC levels were estimated by labeling antisense-treated or nontreated myotubes with NHS-biotin and by further analyzing streptavidin agarose-bound (B) and -unbound (U) fractions by immunoblot assay with anti-GRC (bottom). (b and c) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch-induced CK efflux from antisense-treated or nontreated myotubes. Other conditions were the same as those in Fig. 3 (b and c). Error bars show means ± SD and asterisks show P

    Journal: The Journal of Cell Biology

    Article Title: A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel

    doi: 10.1083/jcb.200301101

    Figure Lengend Snippet: Effect of GRC antisense expression on BIO14.6 myotubes. (a) Immunoblot assay for GRC and β-dystroglycan (β-DG; top) and GRC immunohistochemistry (middle) of BIO14.6 myotubes infected with Ad.β-gal or Ad-antisense–GRC cDNA (Ad.asGRC). Cell surface GRC levels were estimated by labeling antisense-treated or nontreated myotubes with NHS-biotin and by further analyzing streptavidin agarose-bound (B) and -unbound (U) fractions by immunoblot assay with anti-GRC (bottom). (b and c) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch-induced CK efflux from antisense-treated or nontreated myotubes. Other conditions were the same as those in Fig. 3 (b and c). Error bars show means ± SD and asterisks show P

    Article Snippet: NHS-biotin and streptavidin-conjugated agarose were purchased from Pierce Chemical Co. Fluo-4AM was purchased from Molecular Probes and fura-2AM was purchased from Dojin Chemicals.

    Techniques: Expressing, Immunohistochemistry, Infection, Labeling, Fluorescence

    BAX Interaction Site Analysis by PUMA SAHB Photoaffinity Labeling and Mass Spectrometry (A) PUMA pSAHB-1 was incubated with full-length BAX and the mixture subjected to UV irradiation, streptavidin pulldown, electrophoresis, excision of the crosslinked protein, trypsin proteolysis, and LC-MS/MS analysis. A single arginine substitution (A150R) was made in PUMA pSAHB-1 to facilitate tryptic digestion into shorter and more identifiable fragments by MS. The plot (left) depicts the frequency of crosslinked sites identified across the BAX polypeptide sequence, with crosslinked residues mapped onto the solution structure of monomeric BAX (PDB ID 2K7W) (right) and colored according to the frequency of occurrence. The C-terminal α9 helix of BAX has been removed from the structure to better visualize the crosslinked residues of the canonical BH3-binding pocket. (B–D) The corresponding analysis was performed for PUMA pSAHB-2 with full length BAX (B), PUMA pSAHB-1 with C-terminal helix-deleted BAX (BAXΔC) (C), and PUMA pSAHB-2 with BAXΔC (D). X, stapling amino acid; B, norleucine; U, 4-benzoyl-phenylalanine (Bpa). .

    Journal: Chemistry & biology

    Article Title: Multimodal Interaction with BCL-2 Family Proteins Underlies the Pro-Apoptotic Activity of PUMA BH3

    doi: 10.1016/j.chembiol.2013.06.007

    Figure Lengend Snippet: BAX Interaction Site Analysis by PUMA SAHB Photoaffinity Labeling and Mass Spectrometry (A) PUMA pSAHB-1 was incubated with full-length BAX and the mixture subjected to UV irradiation, streptavidin pulldown, electrophoresis, excision of the crosslinked protein, trypsin proteolysis, and LC-MS/MS analysis. A single arginine substitution (A150R) was made in PUMA pSAHB-1 to facilitate tryptic digestion into shorter and more identifiable fragments by MS. The plot (left) depicts the frequency of crosslinked sites identified across the BAX polypeptide sequence, with crosslinked residues mapped onto the solution structure of monomeric BAX (PDB ID 2K7W) (right) and colored according to the frequency of occurrence. The C-terminal α9 helix of BAX has been removed from the structure to better visualize the crosslinked residues of the canonical BH3-binding pocket. (B–D) The corresponding analysis was performed for PUMA pSAHB-2 with full length BAX (B), PUMA pSAHB-1 with C-terminal helix-deleted BAX (BAXΔC) (C), and PUMA pSAHB-2 with BAXΔC (D). X, stapling amino acid; B, norleucine; U, 4-benzoyl-phenylalanine (Bpa). .

    Article Snippet: Biotinylated species were captured by incubation with high-capacity streptavidin agarose (Thermo) for 2 hours at 4°C.

    Techniques: Labeling, Mass Spectrometry, Incubation, Irradiation, Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Sequencing, Binding Assay

    RUSH assay to analyze trafficking of SBP‐GFP‐GluK2 in HEK293T cells Schematic representation of the RUSH Cargo Sorting Assay using confocal microscopy. HEK293T cells were transfected with the SBP‐GFP‐GluK2 plasmid that expresses a fusion protein consisting of streptavidin binding peptide (SBP) followed by GFP and GluK2, and a streptavidin‐KDEL “anchor.” The ER retention signal KDEL retains SBP‐containing proteins in the ER. Upon addition of biotin to the cell culture medium, SBP‐GFP‐Gluk2 is released allowing its trafficking through the secretory pathway. The constitutively ER‐retained SEZ6 mutant SEZ6ΔcytoER‐HA (B) and full‐length wild‐type SEZ6FL‐HA (C) were co‐transfected with SBP‐GFP‐GluK2. Biotin was added to elicit release of SBP‐GFP‐GluK2 from the ER (0 min), and cells were fixed at different time points (0, 20, and 40 min). HA‐tagged SEZ6FL and SEZ6ΔcytoER transgenes were labeled with anti‐HA (mouse) and anti‐mouse‐Alexa594 antibodies. Size bars represent 5 μm. Scatter dot plot represents number of vesicles per cell in SBP‐GFP‐GluK2 RUSH experiment with SEZ6ΔcytoER‐HA (control) or SEZ6FL‐HA co‐transfection. Vesicle counts from 15 to 28 cells per timepoint from 2 independent experiments with mean number of vesicles and error bars (SD) are shown. Mann–Whitney test was used to compare HA‐tagged SEZ6FL and SEZ6ΔcytoER at each time point. At 20 min *** P ‐value = 0.0007, at 40 min **** P ‐value

    Journal: The EMBO Journal

    Article Title: Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3

    doi: 10.15252/embj.2019103457

    Figure Lengend Snippet: RUSH assay to analyze trafficking of SBP‐GFP‐GluK2 in HEK293T cells Schematic representation of the RUSH Cargo Sorting Assay using confocal microscopy. HEK293T cells were transfected with the SBP‐GFP‐GluK2 plasmid that expresses a fusion protein consisting of streptavidin binding peptide (SBP) followed by GFP and GluK2, and a streptavidin‐KDEL “anchor.” The ER retention signal KDEL retains SBP‐containing proteins in the ER. Upon addition of biotin to the cell culture medium, SBP‐GFP‐Gluk2 is released allowing its trafficking through the secretory pathway. The constitutively ER‐retained SEZ6 mutant SEZ6ΔcytoER‐HA (B) and full‐length wild‐type SEZ6FL‐HA (C) were co‐transfected with SBP‐GFP‐GluK2. Biotin was added to elicit release of SBP‐GFP‐GluK2 from the ER (0 min), and cells were fixed at different time points (0, 20, and 40 min). HA‐tagged SEZ6FL and SEZ6ΔcytoER transgenes were labeled with anti‐HA (mouse) and anti‐mouse‐Alexa594 antibodies. Size bars represent 5 μm. Scatter dot plot represents number of vesicles per cell in SBP‐GFP‐GluK2 RUSH experiment with SEZ6ΔcytoER‐HA (control) or SEZ6FL‐HA co‐transfection. Vesicle counts from 15 to 28 cells per timepoint from 2 independent experiments with mean number of vesicles and error bars (SD) are shown. Mann–Whitney test was used to compare HA‐tagged SEZ6FL and SEZ6ΔcytoER at each time point. At 20 min *** P ‐value = 0.0007, at 40 min **** P ‐value

    Article Snippet: Cell lysates of three control (GFP) and three SEZ6KO (CRE) were loaded on a polyprep chromatography column (Bio‐Rad) containing 300 μl of high‐capacity streptavidin agarose beads (Thermofisher) and washed with 10 ml 2% SDS in PBS to remove non‐specifically bound proteins.

    Techniques: Confocal Microscopy, Transfection, Plasmid Preparation, Binding Assay, Cell Culture, Mutagenesis, Labeling, Cotransfection, MANN-WHITNEY

    Quality control of surface enrichment by Sulfo‐ NHS ‐Biotin SEZ6KO and WT neurons were biotinylated with Sulfo‐NHS‐Biotin, and surface proteins were enriched by streptavidin bead pull‐down. Total proteins in the lysates (11 μg, “Total”) and surface proteins (60 μg, “Surface”) were analyzed by immunoblotting. The efficiency of the enrichment is shown by the calnexin depletion, by the absence of immature SEZ6 (black star, Pigoni et al , 2016 ) and by the absence of a second GluA2 band at a lower molecular weight in the surface pull‐down compared to the total lysates. GluK2/3 was quantified both in total lysates and at the cell surface, and GluK2/3 surface/total ratio was normalized for SEZ6L2 surface/total (plot shows mean ± SEM, 6 WT, and 7 SEZ6KO replicates and Mann–Whitney test were used. Exact * P ‐value = 0.047). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3

    doi: 10.15252/embj.2019103457

    Figure Lengend Snippet: Quality control of surface enrichment by Sulfo‐ NHS ‐Biotin SEZ6KO and WT neurons were biotinylated with Sulfo‐NHS‐Biotin, and surface proteins were enriched by streptavidin bead pull‐down. Total proteins in the lysates (11 μg, “Total”) and surface proteins (60 μg, “Surface”) were analyzed by immunoblotting. The efficiency of the enrichment is shown by the calnexin depletion, by the absence of immature SEZ6 (black star, Pigoni et al , 2016 ) and by the absence of a second GluA2 band at a lower molecular weight in the surface pull‐down compared to the total lysates. GluK2/3 was quantified both in total lysates and at the cell surface, and GluK2/3 surface/total ratio was normalized for SEZ6L2 surface/total (plot shows mean ± SEM, 6 WT, and 7 SEZ6KO replicates and Mann–Whitney test were used. Exact * P ‐value = 0.047). Source data are available online for this figure.

    Article Snippet: Cell lysates of three control (GFP) and three SEZ6KO (CRE) were loaded on a polyprep chromatography column (Bio‐Rad) containing 300 μl of high‐capacity streptavidin agarose beads (Thermofisher) and washed with 10 ml 2% SDS in PBS to remove non‐specifically bound proteins.

    Techniques: Molecular Weight, MANN-WHITNEY

    Validation of GluK2/3 reduction on the surface of SEZ6KO neurons by immunoblot SEZ6KO and WT neurons were biotinylated with Sulfo‐NHS‐Biotin, and surface proteins were enriched by streptavidin bead pull‐down. The GluK2/3 antibody cannot discriminate the subunits 2 and 3 (Lerma Marques, 2013 ); therefore, the band is commonly indicated with the labeling GluK2/3. GluK2/3, GluA2, and GluN2B surface levels were quantified, normalized to SEZ6L2 surface levels (negative control) in the same sample (GluK2/3/SEZ6L2) and divided by the WT levels, with the ratio for WT being set to 1.0 (plot shows mean ± SEM, at least 10 replicates in 4 independent biological experiments, Mann–Whitney **** P ‐value

    Journal: The EMBO Journal

    Article Title: Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3

    doi: 10.15252/embj.2019103457

    Figure Lengend Snippet: Validation of GluK2/3 reduction on the surface of SEZ6KO neurons by immunoblot SEZ6KO and WT neurons were biotinylated with Sulfo‐NHS‐Biotin, and surface proteins were enriched by streptavidin bead pull‐down. The GluK2/3 antibody cannot discriminate the subunits 2 and 3 (Lerma Marques, 2013 ); therefore, the band is commonly indicated with the labeling GluK2/3. GluK2/3, GluA2, and GluN2B surface levels were quantified, normalized to SEZ6L2 surface levels (negative control) in the same sample (GluK2/3/SEZ6L2) and divided by the WT levels, with the ratio for WT being set to 1.0 (plot shows mean ± SEM, at least 10 replicates in 4 independent biological experiments, Mann–Whitney **** P ‐value

    Article Snippet: Cell lysates of three control (GFP) and three SEZ6KO (CRE) were loaded on a polyprep chromatography column (Bio‐Rad) containing 300 μl of high‐capacity streptavidin agarose beads (Thermofisher) and washed with 10 ml 2% SDS in PBS to remove non‐specifically bound proteins.

    Techniques: Labeling, Negative Control, MANN-WHITNEY

    Discovery of peroxiredoxin 6 (PRDX6) as a candidate biomarker for brain injury. Rat brain proteome was fractionated by two-dimensional gel electrophoresis and transferred to polyvinylidene difluoride (PVDF). Blots were probed with serum from control and traumatic brain injury (TBI) rats (1:250), and visualized by enhanced chemiluminescence using pooled anti-rat immunoglobulin G (IgG) and IgM detection antibodies ( A and B , respectively). A feature showing enhanced autoreactivity following TBI (circles) was mapped to a replicate protein gel (C) and identified by peptide mass fingerprinting as PRDX6 (D) . (E) A representative standard curve for the sandwich immunosorbent electrochemiluminescence assay (IEA) developed to measure PRDX6 in human samples. The data reflect findings involving the independent two-dimensional gel analysis of six different pools of control and TBI serum performed in duplicate. Autoimmune signals were mapped to PRDX6 in the duplicate analyses of three of the six pools.

    Journal: Journal of Neurotrauma

    Article Title: Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker

    doi: 10.1089/neu.2014.3736

    Figure Lengend Snippet: Discovery of peroxiredoxin 6 (PRDX6) as a candidate biomarker for brain injury. Rat brain proteome was fractionated by two-dimensional gel electrophoresis and transferred to polyvinylidene difluoride (PVDF). Blots were probed with serum from control and traumatic brain injury (TBI) rats (1:250), and visualized by enhanced chemiluminescence using pooled anti-rat immunoglobulin G (IgG) and IgM detection antibodies ( A and B , respectively). A feature showing enhanced autoreactivity following TBI (circles) was mapped to a replicate protein gel (C) and identified by peptide mass fingerprinting as PRDX6 (D) . (E) A representative standard curve for the sandwich immunosorbent electrochemiluminescence assay (IEA) developed to measure PRDX6 in human samples. The data reflect findings involving the independent two-dimensional gel analysis of six different pools of control and TBI serum performed in duplicate. Autoimmune signals were mapped to PRDX6 in the duplicate analyses of three of the six pools.

    Article Snippet: A mouse anti-PRDX6 monoclonal antibody (Clone 1A11, GenWay Biotech, catalog #20-007-280008, San Diego, California) and a rabbit monoclonal anti-PRDX6 antibody (Clone EPR3755; Epitomics, catalog # 2769-1, Burlingame, California) were selected for optimization as capture and primary antibodies, respectively.

    Techniques: Biomarker Assay, Two-Dimensional Gel Electrophoresis, Electrophoresis, Peptide Mass Fingerprinting, Electrochemiluminescence

    Peroxiredoxin 6 (PRDX6) is highly expressed in astrocytes in rat cerebral cortex and up-regulated following traumatic brain injury (TBI). Data show expression of PRDX6, glial fibrillary acidic protein (GFAP) and their co-localization in astrocytes in normal rat brain (A, B, C) and TBI rat brain (D, E, F) . PRDX6-expressing astrocytes in the penumbra were enlarged, and more abundant and intensely stained, compared with those more distant from the injury. Findings presented are representative of six independent analyses. PRDX6 expression in neurons and microglia was very low (not shown). Scale bar: 250 μm. Color image is available online at www.liebertpub.com/neu

    Journal: Journal of Neurotrauma

    Article Title: Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker

    doi: 10.1089/neu.2014.3736

    Figure Lengend Snippet: Peroxiredoxin 6 (PRDX6) is highly expressed in astrocytes in rat cerebral cortex and up-regulated following traumatic brain injury (TBI). Data show expression of PRDX6, glial fibrillary acidic protein (GFAP) and their co-localization in astrocytes in normal rat brain (A, B, C) and TBI rat brain (D, E, F) . PRDX6-expressing astrocytes in the penumbra were enlarged, and more abundant and intensely stained, compared with those more distant from the injury. Findings presented are representative of six independent analyses. PRDX6 expression in neurons and microglia was very low (not shown). Scale bar: 250 μm. Color image is available online at www.liebertpub.com/neu

    Article Snippet: A mouse anti-PRDX6 monoclonal antibody (Clone 1A11, GenWay Biotech, catalog #20-007-280008, San Diego, California) and a rabbit monoclonal anti-PRDX6 antibody (Clone EPR3755; Epitomics, catalog # 2769-1, Burlingame, California) were selected for optimization as capture and primary antibodies, respectively.

    Techniques: Expressing, Staining

    Comparison of levels of peroxiredoxin 6 (PRDX6) in human plasma and serum. Serum and plasma were prepared as matched sets from blood samples drawn from normal male and female volunteers ( n =10 each). Levels of PRDX6 were measured by immunosorbent electrochemiluminescence assay (IEA). The experiment was replicated in a second, independent cohort of the same size (A, B) . Differences with statistical significance are shown by the arrows and corresponding p values.

    Journal: Journal of Neurotrauma

    Article Title: Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker

    doi: 10.1089/neu.2014.3736

    Figure Lengend Snippet: Comparison of levels of peroxiredoxin 6 (PRDX6) in human plasma and serum. Serum and plasma were prepared as matched sets from blood samples drawn from normal male and female volunteers ( n =10 each). Levels of PRDX6 were measured by immunosorbent electrochemiluminescence assay (IEA). The experiment was replicated in a second, independent cohort of the same size (A, B) . Differences with statistical significance are shown by the arrows and corresponding p values.

    Article Snippet: A mouse anti-PRDX6 monoclonal antibody (Clone 1A11, GenWay Biotech, catalog #20-007-280008, San Diego, California) and a rabbit monoclonal anti-PRDX6 antibody (Clone EPR3755; Epitomics, catalog # 2769-1, Burlingame, California) were selected for optimization as capture and primary antibodies, respectively.

    Techniques: Electrochemiluminescence

    Western blot analysis of peroxiredoxin 6 (PRDX6) in extracts of human brain and platelets. Recombinant PRDX6 (200 ng, lane 1), human brain extract (20 μg, and 10 μg; lanes 2 and 3, respectively) and human platelet extract (20 μg, 10 μg, 5 μg, and 2.5 μg; lanes 4-7, respectively) were analyzed by silver staining (A) and western blot and probed with anti-PRDX6 antibody (B) . The recombinant PRDX6 standard exhibited a higher molecular weight, compared with tissue PRDX6 due to the presence of a histidine tag.

    Journal: Journal of Neurotrauma

    Article Title: Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker

    doi: 10.1089/neu.2014.3736

    Figure Lengend Snippet: Western blot analysis of peroxiredoxin 6 (PRDX6) in extracts of human brain and platelets. Recombinant PRDX6 (200 ng, lane 1), human brain extract (20 μg, and 10 μg; lanes 2 and 3, respectively) and human platelet extract (20 μg, 10 μg, 5 μg, and 2.5 μg; lanes 4-7, respectively) were analyzed by silver staining (A) and western blot and probed with anti-PRDX6 antibody (B) . The recombinant PRDX6 standard exhibited a higher molecular weight, compared with tissue PRDX6 due to the presence of a histidine tag.

    Article Snippet: A mouse anti-PRDX6 monoclonal antibody (Clone 1A11, GenWay Biotech, catalog #20-007-280008, San Diego, California) and a rabbit monoclonal anti-PRDX6 antibody (Clone EPR3755; Epitomics, catalog # 2769-1, Burlingame, California) were selected for optimization as capture and primary antibodies, respectively.

    Techniques: Western Blot, Recombinant, Silver Staining, Molecular Weight

    Expression of the ∼65-kDa E-selectin ligand is characteristic of MLs and circulating myeloid cells during leukemoid reactions (FL), and comprises the heavy chain of MPO. ( A ) Lysates of mobilized leukocytes (MLs), native leukocytes (NLs), and circulating myeloid cells of patients with febrile leukocytosis (FL), normalized for input cell number, were resolved by SDS/PAGE and stained in Western blot with E-selectin Ig chimera (E–Ig). A representative blot of multiple patient samples ( n > 14 for each group) is shown. Expression of the ∼65-kDa glycoprotein is characteristic of G-CSF–primed cells (MLs and FLs), but not of NLs. ( B ) Lysates of MLs (lane 1) and WGA lectin chromatography-purified glycoproteins (lane 2) resolved on reducing 7.5% SDS/PAGE gel and immunoblotted with E-selectin–Ig chimera. E-selectin ligands are present at ∼140 kDa (PSGL-1), ∼100 kDa (HCELL), and at ∼65 kDa. ( C ) Representative results of Western blots of MPO immunoprecipitates (IPs) from MLs resolved under nonreducing (lane 1) or reducing conditions (lanes 2 and 3) and stained with anti-MPO mAb. In nonreduced gels, the mature homodimer of ∼140 kDa is evident (lane 1). Under reducing conditions, Western blot with anti-MPO mAb 2C7 (lane 2) reveals bands at ∼90 kDa (precursor) and ∼65 kDa (heavy chain) or only the ∼65-kDa band when stained with anti-MPO mAb 3D3 (lane 3).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: G-CSF Induces Membrane Expression of a Myeloperoxidase Glycovariant that Operates as an E-selectin Ligand on Human Myeloid Cells

    doi: 10.1073/pnas.1320833111

    Figure Lengend Snippet: Expression of the ∼65-kDa E-selectin ligand is characteristic of MLs and circulating myeloid cells during leukemoid reactions (FL), and comprises the heavy chain of MPO. ( A ) Lysates of mobilized leukocytes (MLs), native leukocytes (NLs), and circulating myeloid cells of patients with febrile leukocytosis (FL), normalized for input cell number, were resolved by SDS/PAGE and stained in Western blot with E-selectin Ig chimera (E–Ig). A representative blot of multiple patient samples ( n > 14 for each group) is shown. Expression of the ∼65-kDa glycoprotein is characteristic of G-CSF–primed cells (MLs and FLs), but not of NLs. ( B ) Lysates of MLs (lane 1) and WGA lectin chromatography-purified glycoproteins (lane 2) resolved on reducing 7.5% SDS/PAGE gel and immunoblotted with E-selectin–Ig chimera. E-selectin ligands are present at ∼140 kDa (PSGL-1), ∼100 kDa (HCELL), and at ∼65 kDa. ( C ) Representative results of Western blots of MPO immunoprecipitates (IPs) from MLs resolved under nonreducing (lane 1) or reducing conditions (lanes 2 and 3) and stained with anti-MPO mAb. In nonreduced gels, the mature homodimer of ∼140 kDa is evident (lane 1). Under reducing conditions, Western blot with anti-MPO mAb 2C7 (lane 2) reveals bands at ∼90 kDa (precursor) and ∼65 kDa (heavy chain) or only the ∼65-kDa band when stained with anti-MPO mAb 3D3 (lane 3).

    Article Snippet: Biochemical analysis showed that this augmented endothelial adherence of G-CSF–mobilized human leukocytes (MLs) was due to increased E-selectin ligand activity, in part mediated by expression of a novel E-selectin ligand, a membrane glycoprotein with a molecular weight of ∼65 kDa ( ).

    Techniques: Expressing, SDS Page, Staining, Western Blot, Whole Genome Amplification, Chromatography, Purification

    G-CSF induces cell surface expression and E-selectin ligand activity of MPO. ( A ) MPO immunoprecipitates (IPs) from lysates of bone marrow myeloid cells (BMs), NLs, and MLs stained in Western blot with anti-MPO mAb clone 3D3. ( B ) MPO IPs resolved on reducing SDS/PAGE and stained with E-selectin Ig chimera. ( C ) Flow cytometry analysis of NLs, BMs, and MLs cultured for 48 h without G-CSF (−) or with G-CSF (+). Values represent mean ± SD of percent MPO-positive cells from multiple donors ( n > 30), * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: G-CSF Induces Membrane Expression of a Myeloperoxidase Glycovariant that Operates as an E-selectin Ligand on Human Myeloid Cells

    doi: 10.1073/pnas.1320833111

    Figure Lengend Snippet: G-CSF induces cell surface expression and E-selectin ligand activity of MPO. ( A ) MPO immunoprecipitates (IPs) from lysates of bone marrow myeloid cells (BMs), NLs, and MLs stained in Western blot with anti-MPO mAb clone 3D3. ( B ) MPO IPs resolved on reducing SDS/PAGE and stained with E-selectin Ig chimera. ( C ) Flow cytometry analysis of NLs, BMs, and MLs cultured for 48 h without G-CSF (−) or with G-CSF (+). Values represent mean ± SD of percent MPO-positive cells from multiple donors ( n > 30), * P

    Article Snippet: Biochemical analysis showed that this augmented endothelial adherence of G-CSF–mobilized human leukocytes (MLs) was due to increased E-selectin ligand activity, in part mediated by expression of a novel E-selectin ligand, a membrane glycoprotein with a molecular weight of ∼65 kDa ( ).

    Techniques: Expressing, Activity Assay, Staining, Western Blot, SDS Page, Flow Cytometry, Cytometry, Cell Culture

    Inhibition of E-selectin receptor/ligand interactions and of MPO activity suppresses G-CSF–induced myeloid cell angiotoxicity. ( A ) Spectrophotometric detection of membrane MPO activity from NLs cultured in the presence or absence of G-CSF and DMJ. Values represent the relative change in absorbance with respect to substrate alone ( n = 5), * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: G-CSF Induces Membrane Expression of a Myeloperoxidase Glycovariant that Operates as an E-selectin Ligand on Human Myeloid Cells

    doi: 10.1073/pnas.1320833111

    Figure Lengend Snippet: Inhibition of E-selectin receptor/ligand interactions and of MPO activity suppresses G-CSF–induced myeloid cell angiotoxicity. ( A ) Spectrophotometric detection of membrane MPO activity from NLs cultured in the presence or absence of G-CSF and DMJ. Values represent the relative change in absorbance with respect to substrate alone ( n = 5), * P

    Article Snippet: Biochemical analysis showed that this augmented endothelial adherence of G-CSF–mobilized human leukocytes (MLs) was due to increased E-selectin ligand activity, in part mediated by expression of a novel E-selectin ligand, a membrane glycoprotein with a molecular weight of ∼65 kDa ( ).

    Techniques: Inhibition, Activity Assay, Cell Culture

    Discovery of peroxiredoxin 6 (PRDX6) as a candidate biomarker for brain injury. Rat brain proteome was fractionated by two-dimensional gel electrophoresis and transferred to polyvinylidene difluoride (PVDF). Blots were probed with serum from control and traumatic brain injury (TBI) rats (1:250), and visualized by enhanced chemiluminescence using pooled anti-rat immunoglobulin G (IgG) and IgM detection antibodies ( A and B , respectively). A feature showing enhanced autoreactivity following TBI (circles) was mapped to a replicate protein gel (C) and identified by peptide mass fingerprinting as PRDX6 (D) . (E) A representative standard curve for the sandwich immunosorbent electrochemiluminescence assay (IEA) developed to measure PRDX6 in human samples. The data reflect findings involving the independent two-dimensional gel analysis of six different pools of control and TBI serum performed in duplicate. Autoimmune signals were mapped to PRDX6 in the duplicate analyses of three of the six pools.

    Journal: Journal of Neurotrauma

    Article Title: Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker

    doi: 10.1089/neu.2014.3736

    Figure Lengend Snippet: Discovery of peroxiredoxin 6 (PRDX6) as a candidate biomarker for brain injury. Rat brain proteome was fractionated by two-dimensional gel electrophoresis and transferred to polyvinylidene difluoride (PVDF). Blots were probed with serum from control and traumatic brain injury (TBI) rats (1:250), and visualized by enhanced chemiluminescence using pooled anti-rat immunoglobulin G (IgG) and IgM detection antibodies ( A and B , respectively). A feature showing enhanced autoreactivity following TBI (circles) was mapped to a replicate protein gel (C) and identified by peptide mass fingerprinting as PRDX6 (D) . (E) A representative standard curve for the sandwich immunosorbent electrochemiluminescence assay (IEA) developed to measure PRDX6 in human samples. The data reflect findings involving the independent two-dimensional gel analysis of six different pools of control and TBI serum performed in duplicate. Autoimmune signals were mapped to PRDX6 in the duplicate analyses of three of the six pools.

    Article Snippet: A mouse anti-PRDX6 monoclonal antibody (Clone 1A11, GenWay Biotech, catalog #20-007-280008, San Diego, California) and a rabbit monoclonal anti-PRDX6 antibody (Clone EPR3755; Epitomics, catalog # 2769-1, Burlingame, California) were selected for optimization as capture and primary antibodies, respectively.

    Techniques: Biomarker Assay, Two-Dimensional Gel Electrophoresis, Electrophoresis, Peptide Mass Fingerprinting, Electrochemiluminescence

    Peroxiredoxin 6 (PRDX6) is highly expressed in astrocytes in rat cerebral cortex and up-regulated following traumatic brain injury (TBI). Data show expression of PRDX6, glial fibrillary acidic protein (GFAP) and their co-localization in astrocytes in normal rat brain (A, B, C) and TBI rat brain (D, E, F) . PRDX6-expressing astrocytes in the penumbra were enlarged, and more abundant and intensely stained, compared with those more distant from the injury. Findings presented are representative of six independent analyses. PRDX6 expression in neurons and microglia was very low (not shown). Scale bar: 250 μm. Color image is available online at www.liebertpub.com/neu

    Journal: Journal of Neurotrauma

    Article Title: Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker

    doi: 10.1089/neu.2014.3736

    Figure Lengend Snippet: Peroxiredoxin 6 (PRDX6) is highly expressed in astrocytes in rat cerebral cortex and up-regulated following traumatic brain injury (TBI). Data show expression of PRDX6, glial fibrillary acidic protein (GFAP) and their co-localization in astrocytes in normal rat brain (A, B, C) and TBI rat brain (D, E, F) . PRDX6-expressing astrocytes in the penumbra were enlarged, and more abundant and intensely stained, compared with those more distant from the injury. Findings presented are representative of six independent analyses. PRDX6 expression in neurons and microglia was very low (not shown). Scale bar: 250 μm. Color image is available online at www.liebertpub.com/neu

    Article Snippet: A mouse anti-PRDX6 monoclonal antibody (Clone 1A11, GenWay Biotech, catalog #20-007-280008, San Diego, California) and a rabbit monoclonal anti-PRDX6 antibody (Clone EPR3755; Epitomics, catalog # 2769-1, Burlingame, California) were selected for optimization as capture and primary antibodies, respectively.

    Techniques: Expressing, Staining

    Comparison of levels of peroxiredoxin 6 (PRDX6) in human plasma and serum. Serum and plasma were prepared as matched sets from blood samples drawn from normal male and female volunteers ( n =10 each). Levels of PRDX6 were measured by immunosorbent electrochemiluminescence assay (IEA). The experiment was replicated in a second, independent cohort of the same size (A, B) . Differences with statistical significance are shown by the arrows and corresponding p values.

    Journal: Journal of Neurotrauma

    Article Title: Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker

    doi: 10.1089/neu.2014.3736

    Figure Lengend Snippet: Comparison of levels of peroxiredoxin 6 (PRDX6) in human plasma and serum. Serum and plasma were prepared as matched sets from blood samples drawn from normal male and female volunteers ( n =10 each). Levels of PRDX6 were measured by immunosorbent electrochemiluminescence assay (IEA). The experiment was replicated in a second, independent cohort of the same size (A, B) . Differences with statistical significance are shown by the arrows and corresponding p values.

    Article Snippet: A mouse anti-PRDX6 monoclonal antibody (Clone 1A11, GenWay Biotech, catalog #20-007-280008, San Diego, California) and a rabbit monoclonal anti-PRDX6 antibody (Clone EPR3755; Epitomics, catalog # 2769-1, Burlingame, California) were selected for optimization as capture and primary antibodies, respectively.

    Techniques: Electrochemiluminescence

    Western blot analysis of peroxiredoxin 6 (PRDX6) in extracts of human brain and platelets. Recombinant PRDX6 (200 ng, lane 1), human brain extract (20 μg, and 10 μg; lanes 2 and 3, respectively) and human platelet extract (20 μg, 10 μg, 5 μg, and 2.5 μg; lanes 4-7, respectively) were analyzed by silver staining (A) and western blot and probed with anti-PRDX6 antibody (B) . The recombinant PRDX6 standard exhibited a higher molecular weight, compared with tissue PRDX6 due to the presence of a histidine tag.

    Journal: Journal of Neurotrauma

    Article Title: Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker

    doi: 10.1089/neu.2014.3736

    Figure Lengend Snippet: Western blot analysis of peroxiredoxin 6 (PRDX6) in extracts of human brain and platelets. Recombinant PRDX6 (200 ng, lane 1), human brain extract (20 μg, and 10 μg; lanes 2 and 3, respectively) and human platelet extract (20 μg, 10 μg, 5 μg, and 2.5 μg; lanes 4-7, respectively) were analyzed by silver staining (A) and western blot and probed with anti-PRDX6 antibody (B) . The recombinant PRDX6 standard exhibited a higher molecular weight, compared with tissue PRDX6 due to the presence of a histidine tag.

    Article Snippet: A mouse anti-PRDX6 monoclonal antibody (Clone 1A11, GenWay Biotech, catalog #20-007-280008, San Diego, California) and a rabbit monoclonal anti-PRDX6 antibody (Clone EPR3755; Epitomics, catalog # 2769-1, Burlingame, California) were selected for optimization as capture and primary antibodies, respectively.

    Techniques: Western Blot, Recombinant, Silver Staining, Molecular Weight

    Identification of proteins in fraction III. A. MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. B. Immunoblot analysis of fraction III probed with anti-PAP antibody. Molecular weight markers shown are in kDa.

    Journal: PLoS ONE

    Article Title: Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

    doi: 10.1371/journal.pone.0058631

    Figure Lengend Snippet: Identification of proteins in fraction III. A. MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. B. Immunoblot analysis of fraction III probed with anti-PAP antibody. Molecular weight markers shown are in kDa.

    Article Snippet: Fraction III (20 µg) was then resolved on a 12.5% SDS-PAGE and subjected to immunoblot analysis using mouse monoclonal anti-PAP antibody (US Biological, Swampscott, MA), at 1∶2000 dilution in TBS-T buffer, for 1 h at RT.

    Techniques: High Performance Liquid Chromatography, Molecular Weight

    Detection of PSP94-PAP complex in human seminal plasma. A. PSP94-PAP complex from seminal plasma (100 µg) was co-immunoprecipitated with anti-PAP antibody (lane 1). Protein G beads incubated with seminal plasma in buffer alone served as the negative control (lane 2). 20 µg of seminal plasma was loaded in lane 3 as input and the immunoblot was probed with anti-PSP94 antibody. The immunoreactive band of PSP94 (∼17 kDa) is detected only in lane 1 and not in lane 2. B. PSP94-PAP complex from seminal plasma (100 µg) was co-immunoprecipitated with anti-PSP94 antibody (lane 1). Protein G beads incubated with seminal plasma in buffer alone served as the negative control (lane 2). 20 µg of seminal plasma was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. The immunoreactive band of PAP (∼47 kDa) is detected only in lane 1 and not in lane 2. Molecular weight markers shown are in kDa.

    Journal: PLoS ONE

    Article Title: Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

    doi: 10.1371/journal.pone.0058631

    Figure Lengend Snippet: Detection of PSP94-PAP complex in human seminal plasma. A. PSP94-PAP complex from seminal plasma (100 µg) was co-immunoprecipitated with anti-PAP antibody (lane 1). Protein G beads incubated with seminal plasma in buffer alone served as the negative control (lane 2). 20 µg of seminal plasma was loaded in lane 3 as input and the immunoblot was probed with anti-PSP94 antibody. The immunoreactive band of PSP94 (∼17 kDa) is detected only in lane 1 and not in lane 2. B. PSP94-PAP complex from seminal plasma (100 µg) was co-immunoprecipitated with anti-PSP94 antibody (lane 1). Protein G beads incubated with seminal plasma in buffer alone served as the negative control (lane 2). 20 µg of seminal plasma was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. The immunoreactive band of PAP (∼47 kDa) is detected only in lane 1 and not in lane 2. Molecular weight markers shown are in kDa.

    Article Snippet: Fraction III (20 µg) was then resolved on a 12.5% SDS-PAGE and subjected to immunoblot analysis using mouse monoclonal anti-PAP antibody (US Biological, Swampscott, MA), at 1∶2000 dilution in TBS-T buffer, for 1 h at RT.

    Techniques: Immunoprecipitation, Incubation, Negative Control, Molecular Weight

    Identification and characterization of affinity purified PSP94 binding protein from fraction III. A. Gel stained with silver nitrate showing the presence of a band at ∼47 kDa in the eluate lane, further identified to be PAP protein on MS and MS/MS analysis ( Figure S2 ). B. Immunoblot probed with anti-PAP antibody showing a band at ∼47 kDa in eluate (lane 3) corresponding to the band of immunoreactive PAP protein detected in the input (lane 1; 10 µg). The last wash (lane 2) did not show any band. Molecular weight markers shown are in kDa.

    Journal: PLoS ONE

    Article Title: Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

    doi: 10.1371/journal.pone.0058631

    Figure Lengend Snippet: Identification and characterization of affinity purified PSP94 binding protein from fraction III. A. Gel stained with silver nitrate showing the presence of a band at ∼47 kDa in the eluate lane, further identified to be PAP protein on MS and MS/MS analysis ( Figure S2 ). B. Immunoblot probed with anti-PAP antibody showing a band at ∼47 kDa in eluate (lane 3) corresponding to the band of immunoreactive PAP protein detected in the input (lane 1; 10 µg). The last wash (lane 2) did not show any band. Molecular weight markers shown are in kDa.

    Article Snippet: Fraction III (20 µg) was then resolved on a 12.5% SDS-PAGE and subjected to immunoblot analysis using mouse monoclonal anti-PAP antibody (US Biological, Swampscott, MA), at 1∶2000 dilution in TBS-T buffer, for 1 h at RT.

    Techniques: Affinity Purification, Binding Assay, Staining, Mass Spectrometry, Molecular Weight

    Co-immunoprecipitation of PSP94 and PAP proteins from fraction III. A. PSP94-PAP complex from fraction III (50 µg) co-immunoprecipitated with anti-PAP antibody showing the presence of PSP94 (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with mouse isotype control antibody (lane 1) served as controls. 10 µg of fraction III was loaded in lane 4 as input and the immunoblot was probed with anti-PSP94 antibody. B. PSP94-PAP complex from fraction III (50 µg) was co-immunoprecipitated with anti-PSP94 antibody showing the presence of PAP (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with normal rabbit serum (lane 1) served as controls. 10 µg of fraction III was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. Molecular weight markers shown are in kDa.

    Journal: PLoS ONE

    Article Title: Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

    doi: 10.1371/journal.pone.0058631

    Figure Lengend Snippet: Co-immunoprecipitation of PSP94 and PAP proteins from fraction III. A. PSP94-PAP complex from fraction III (50 µg) co-immunoprecipitated with anti-PAP antibody showing the presence of PSP94 (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with mouse isotype control antibody (lane 1) served as controls. 10 µg of fraction III was loaded in lane 4 as input and the immunoblot was probed with anti-PSP94 antibody. B. PSP94-PAP complex from fraction III (50 µg) was co-immunoprecipitated with anti-PSP94 antibody showing the presence of PAP (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with normal rabbit serum (lane 1) served as controls. 10 µg of fraction III was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. Molecular weight markers shown are in kDa.

    Article Snippet: Fraction III (20 µg) was then resolved on a 12.5% SDS-PAGE and subjected to immunoblot analysis using mouse monoclonal anti-PAP antibody (US Biological, Swampscott, MA), at 1∶2000 dilution in TBS-T buffer, for 1 h at RT.

    Techniques: Immunoprecipitation, Incubation, Molecular Weight