hrp conjugated goat anti mouse igg  (thermo fisher)


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    Name:
    Goat anti Mouse IgG H L Poly HRP Secondary Antibody
    Description:
    Goat anti Mouse IgG H L Poly HRP Secondary Antibody for Western Blot IHC ELISA
    Catalog Number:
    32230
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    thermo fisher hrp conjugated goat anti mouse igg
    Immunohistochemical staining of gastric cancer (GC) tissues to verify scFv specificity. Ten biopsies of diffuse and intestinal gastric adenocarcinomas as well as normal gastric slides were stained with F10 and G1 scFv fragments. After deparaffinization and antigen retrieval in prewarmed citrate buffer, tissue sections were sequentially incubated with scFv fragments, anti‐c‐Myc mAb and horseradish peroxidase <t>(HRP)‐conjugated</t> goat anti‐mouse <t>IgG.</t> scFv binding was visualized by 3,3‐diaminobenzidine (DAB) and nuclei were counterstained with haematoxylin. Both F10 and G1 revealed strong membranous/cytoplasmic staining on diffuse gastric adenocarcinoma biopsies without any cross‐reactivity to intestinal gastric adenocarcinoma and normal gastric tissues. Magnification, 400 ×.
    Goat anti Mouse IgG H L Poly HRP Secondary Antibody for Western Blot IHC ELISA
    https://www.bioz.com/result/hrp conjugated goat anti mouse igg/product/thermo fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated goat anti mouse igg - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "Tailoring subtractive cell biopanning to identify diffuse gastric adenocarcinoma‐associated antigens via human scFv antibodies"

    Article Title: Tailoring subtractive cell biopanning to identify diffuse gastric adenocarcinoma‐associated antigens via human scFv antibodies

    Journal: Immunology

    doi: 10.1111/imm.13129

    Immunohistochemical staining of gastric cancer (GC) tissues to verify scFv specificity. Ten biopsies of diffuse and intestinal gastric adenocarcinomas as well as normal gastric slides were stained with F10 and G1 scFv fragments. After deparaffinization and antigen retrieval in prewarmed citrate buffer, tissue sections were sequentially incubated with scFv fragments, anti‐c‐Myc mAb and horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG. scFv binding was visualized by 3,3‐diaminobenzidine (DAB) and nuclei were counterstained with haematoxylin. Both F10 and G1 revealed strong membranous/cytoplasmic staining on diffuse gastric adenocarcinoma biopsies without any cross‐reactivity to intestinal gastric adenocarcinoma and normal gastric tissues. Magnification, 400 ×.
    Figure Legend Snippet: Immunohistochemical staining of gastric cancer (GC) tissues to verify scFv specificity. Ten biopsies of diffuse and intestinal gastric adenocarcinomas as well as normal gastric slides were stained with F10 and G1 scFv fragments. After deparaffinization and antigen retrieval in prewarmed citrate buffer, tissue sections were sequentially incubated with scFv fragments, anti‐c‐Myc mAb and horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG. scFv binding was visualized by 3,3‐diaminobenzidine (DAB) and nuclei were counterstained with haematoxylin. Both F10 and G1 revealed strong membranous/cytoplasmic staining on diffuse gastric adenocarcinoma biopsies without any cross‐reactivity to intestinal gastric adenocarcinoma and normal gastric tissues. Magnification, 400 ×.

    Techniques Used: Immunohistochemistry, Staining, Incubation, Binding Assay

    Sodium dodecyl sulphate−polyacrylamide (SDS−PAGE) and Western blot analysis of expression and purification. The samples related to expression and purification steps were analysed by 12% SDS−PAGE (a, b) followed by immunoblotting (c, d). The gels were stained with Coomassie blue R250. The blots were probed by anti‐c‐Myc mAb and horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG, followed by incubation with ECL and appearing on X‐ray film. The arrows denote the approximate molecular weight of the scFvs. Molecular weight standards in kDa, M; uninduced total cell lysate, UN; induced total cell lysate, IN; uninduced periplasmic extracts, UNP; induced periplasmic extracts related to the selected clones; periplasmic extraction, P; flow‐through, F; wash fraction, W; eluted fractions of each scFv clones.
    Figure Legend Snippet: Sodium dodecyl sulphate−polyacrylamide (SDS−PAGE) and Western blot analysis of expression and purification. The samples related to expression and purification steps were analysed by 12% SDS−PAGE (a, b) followed by immunoblotting (c, d). The gels were stained with Coomassie blue R250. The blots were probed by anti‐c‐Myc mAb and horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG, followed by incubation with ECL and appearing on X‐ray film. The arrows denote the approximate molecular weight of the scFvs. Molecular weight standards in kDa, M; uninduced total cell lysate, UN; induced total cell lysate, IN; uninduced periplasmic extracts, UNP; induced periplasmic extracts related to the selected clones; periplasmic extraction, P; flow‐through, F; wash fraction, W; eluted fractions of each scFv clones.

    Techniques Used: SDS Page, Western Blot, Expressing, Purification, Staining, Incubation, Molecular Weight, Clone Assay

    Binding specificity analysis of the purified scFvs. In ELISA test (a), 5 × 10 5 MKN‐45 (black bars), AGS (stripped bars) and NIH‐3T3 (grey bars) cells were sequentially incubated with purified scFvs (2 µg/well), anti‐c‐Myc mAb and horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG. For all scFvs, absorbance values that represent standard error of the mean from duplicate experiments were in favour of MKN‐45 cells. In flow cytometry analysis (b), MKN‐45, AGS and NIH‐3T3 cells were incubated with purified scFv fragments (2 µg) for 30 min on ice. Bound scFvs were detected by anti‐c‐Myc mAb and fluorescein isothiocyanate (FITC)‐labelled rat anti‐mouse IgG. All plot histograms represent analysis of live cells by excluding PI‐positive cells (dead cells). Fluorescence intensity of the scFv staining (green line) in contrast to the corresponding isotype controls (filled grey peaks) were analysed by FACSCalibur and CellQuest Pro software. All scFvs bind preferentially to MKN‐45 and not to the NIH‐3T3 and AGS (negative control cells). Unrelated scFv was used as negative control.
    Figure Legend Snippet: Binding specificity analysis of the purified scFvs. In ELISA test (a), 5 × 10 5 MKN‐45 (black bars), AGS (stripped bars) and NIH‐3T3 (grey bars) cells were sequentially incubated with purified scFvs (2 µg/well), anti‐c‐Myc mAb and horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG. For all scFvs, absorbance values that represent standard error of the mean from duplicate experiments were in favour of MKN‐45 cells. In flow cytometry analysis (b), MKN‐45, AGS and NIH‐3T3 cells were incubated with purified scFv fragments (2 µg) for 30 min on ice. Bound scFvs were detected by anti‐c‐Myc mAb and fluorescein isothiocyanate (FITC)‐labelled rat anti‐mouse IgG. All plot histograms represent analysis of live cells by excluding PI‐positive cells (dead cells). Fluorescence intensity of the scFv staining (green line) in contrast to the corresponding isotype controls (filled grey peaks) were analysed by FACSCalibur and CellQuest Pro software. All scFvs bind preferentially to MKN‐45 and not to the NIH‐3T3 and AGS (negative control cells). Unrelated scFv was used as negative control.

    Techniques Used: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Fluorescence, Staining, Software, Negative Control

    2) Product Images from "Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies"

    Article Title: Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

    Journal: AMB Express

    doi: 10.1186/s13568-020-01063-x

    Expression and purification analysis. a Shows a sodium dodecyl sulfate–polyacrylamide gel (SDS − PAGE) analysis to confirm the expression of anti-G17-Gly. The identical amount of samples including UN, uninduced total cell lysate; IN, induced total cell lysate; UNP, uninduced periplasm fraction; and INP, induced periplasmic fraction were separated on 12% SDS-PAGE and then stained with Coomassie Brilliant Blue R250. b , c Show confirmation of the purification process by 12% SDS-PAGE and immunoblotting, respectively. The blots were probed by anti‐c‐myc mAb (9E10) and horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG, consecutively. The protein bands were appeared on X‐ray film by incubation with ECL substrate. The arrowheads denote the approximate molecular weight of the scFvs (around 28 kDa). M Molecular weight standards in kDa, P periplasmic extraction, F flow‐through, E1 and E2 eluted fractions
    Figure Legend Snippet: Expression and purification analysis. a Shows a sodium dodecyl sulfate–polyacrylamide gel (SDS − PAGE) analysis to confirm the expression of anti-G17-Gly. The identical amount of samples including UN, uninduced total cell lysate; IN, induced total cell lysate; UNP, uninduced periplasm fraction; and INP, induced periplasmic fraction were separated on 12% SDS-PAGE and then stained with Coomassie Brilliant Blue R250. b , c Show confirmation of the purification process by 12% SDS-PAGE and immunoblotting, respectively. The blots were probed by anti‐c‐myc mAb (9E10) and horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG, consecutively. The protein bands were appeared on X‐ray film by incubation with ECL substrate. The arrowheads denote the approximate molecular weight of the scFvs (around 28 kDa). M Molecular weight standards in kDa, P periplasmic extraction, F flow‐through, E1 and E2 eluted fractions

    Techniques Used: Expressing, Purification, SDS Page, Staining, Incubation, Molecular Weight

    3) Product Images from "Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein *"

    Article Title: Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.142026

    Binding of CRP to six different immobilized proteins as a function of pH and temperature. Results of a protein ligand-binding assay are shown. Microtiter wells were coated with factor H, oxLDL, C3b, IgG, Aβ, and BSA. The unreacted sites in the wells were blocked with gelatin. CRP (10 μg/ml), diluted in TBS-Ca, pH 7.0–4.6, was then added to the wells. One set of wells was incubated at 25 °C and another at 37 °C, for 2 h. Bound CRP was detected by using a rabbit polyclonal anti-CRP antibody and HRP-conjugated donkey anti-rabbit IgG. The absorbance of the developed color was read at 405 nm and plotted on the y axis.
    Figure Legend Snippet: Binding of CRP to six different immobilized proteins as a function of pH and temperature. Results of a protein ligand-binding assay are shown. Microtiter wells were coated with factor H, oxLDL, C3b, IgG, Aβ, and BSA. The unreacted sites in the wells were blocked with gelatin. CRP (10 μg/ml), diluted in TBS-Ca, pH 7.0–4.6, was then added to the wells. One set of wells was incubated at 25 °C and another at 37 °C, for 2 h. Bound CRP was detected by using a rabbit polyclonal anti-CRP antibody and HRP-conjugated donkey anti-rabbit IgG. The absorbance of the developed color was read at 405 nm and plotted on the y axis.

    Techniques Used: Binding Assay, Ligand Binding Assay, Incubation

    Structural change in CRP after binding to immobilized proteins at acidic pH. Microtiter wells were coated with factor H, oxLDL, C3b, IgG, Aβ, and BSA. CRP (10 μg/ml), diluted in TBS-Ca, pH 7.0–4.6, was then added to the wells and incubated at 37 °C for 2 h. Bound CRP was detected using three different anti-CRP antibodies: a polyclonal anti-CRP antibody, anti-CRP mAb HD2.4, or anti-CRP mAb 3H12. After addition of appropriate HRP-conjugated secondary antibodies, the absorbance of the developed color was read at 405 nm and plotted on the y axis.
    Figure Legend Snippet: Structural change in CRP after binding to immobilized proteins at acidic pH. Microtiter wells were coated with factor H, oxLDL, C3b, IgG, Aβ, and BSA. CRP (10 μg/ml), diluted in TBS-Ca, pH 7.0–4.6, was then added to the wells and incubated at 37 °C for 2 h. Bound CRP was detected using three different anti-CRP antibodies: a polyclonal anti-CRP antibody, anti-CRP mAb HD2.4, or anti-CRP mAb 3H12. After addition of appropriate HRP-conjugated secondary antibodies, the absorbance of the developed color was read at 405 nm and plotted on the y axis.

    Techniques Used: Binding Assay, Incubation

    4) Product Images from "Aminopeptidase N is not required for porcine epidemic diarrhea virus cell entry"

    Article Title: Aminopeptidase N is not required for porcine epidemic diarrhea virus cell entry

    Journal: Virus Research

    doi: 10.1016/j.virusres.2017.03.018

    Cellular overexpression of porcine APN renders MDCK cells susceptible for TGEV, but not for PEDV. (a) MDCK cells stably expressing HA-tagged porcine APN (MDCK-pAPN) were generated and pAPN expression was confirmed by Western blotting with HRP conjugated anti-HA tag antibody (left panel) and immunofluorescence assay (right panel) using mouse anti-HA antibody as primary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) as secondary antibody. A lysate of HEK-293T cells transiently transfected with the HA-tagged pAPN encoding plasmid was taken along as a positive control. Sizes of marker proteins are indicated in kilodaltons. (b) Overexpression of pAPN makes MDCK cells susceptible to TGEV, but not to PEDV. MDCK, MDCK-pAPN and Vero-CCL81 cells were infected with GFP-encoding recombinant PEDV viruses carrying spike proteins of different strains (PEDV strains GDU, CV777 and DR13). The cells were stained with DAPI. The infection experiments were performed three times with similar results, representative images are shown.
    Figure Legend Snippet: Cellular overexpression of porcine APN renders MDCK cells susceptible for TGEV, but not for PEDV. (a) MDCK cells stably expressing HA-tagged porcine APN (MDCK-pAPN) were generated and pAPN expression was confirmed by Western blotting with HRP conjugated anti-HA tag antibody (left panel) and immunofluorescence assay (right panel) using mouse anti-HA antibody as primary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) as secondary antibody. A lysate of HEK-293T cells transiently transfected with the HA-tagged pAPN encoding plasmid was taken along as a positive control. Sizes of marker proteins are indicated in kilodaltons. (b) Overexpression of pAPN makes MDCK cells susceptible to TGEV, but not to PEDV. MDCK, MDCK-pAPN and Vero-CCL81 cells were infected with GFP-encoding recombinant PEDV viruses carrying spike proteins of different strains (PEDV strains GDU, CV777 and DR13). The cells were stained with DAPI. The infection experiments were performed three times with similar results, representative images are shown.

    Techniques Used: Over Expression, Stable Transfection, Expressing, Generated, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation, Positive Control, Marker, Infection, Recombinant, Staining

    5) Product Images from "Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus"

    Article Title: Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21114180

    Identification of the anti-CD4-2 monoclonal antibody (3C8). ( A ) The antibody was screened for specificity and sensitivity against increasing amounts of recombinant CD4-2. Lane 1, rCD3ε at 500 ng; Lane 2, rCD4-1 at 500 ng; Lane 3, rCD4-2 at 16 ng; Lane 4, rCD4-2 at 32 ng; Lane 5, rCD4-2 at 63 ng; Lane 6, rCD4-2 at 125 ng; Lane 7, rCD4-2 at 250 ng; Lane 8, rCD4-2 at 500 ng. ( B ) Reactivity of anti-CD4-2 mAb to transfected 293F cells stably expressing flounder CD4-2 molecules. Scale bar = 20 um. ( C ) Western blotting analysis of leukocyte cell lysates from spleen, head-kidney and peripheral blood of olive flounder using anti-CD4-2 mAb (3C8), followed by HRP-conjugated goat anti-mouse IgG. ( D ) Flow cytometry analysis of head-kidney leukocytes stained with anti-CD4-2-mAb. Negative control; shaded area, Anti-CD4-2-mAb treated; black line. Immunofluorescence staining of head-kidney leukocytes incubated with anti-CD4-2 mAb (3C8), followed by FITC-conjugated goat anti-mouse IgG. Immuno-reactivity appears green.
    Figure Legend Snippet: Identification of the anti-CD4-2 monoclonal antibody (3C8). ( A ) The antibody was screened for specificity and sensitivity against increasing amounts of recombinant CD4-2. Lane 1, rCD3ε at 500 ng; Lane 2, rCD4-1 at 500 ng; Lane 3, rCD4-2 at 16 ng; Lane 4, rCD4-2 at 32 ng; Lane 5, rCD4-2 at 63 ng; Lane 6, rCD4-2 at 125 ng; Lane 7, rCD4-2 at 250 ng; Lane 8, rCD4-2 at 500 ng. ( B ) Reactivity of anti-CD4-2 mAb to transfected 293F cells stably expressing flounder CD4-2 molecules. Scale bar = 20 um. ( C ) Western blotting analysis of leukocyte cell lysates from spleen, head-kidney and peripheral blood of olive flounder using anti-CD4-2 mAb (3C8), followed by HRP-conjugated goat anti-mouse IgG. ( D ) Flow cytometry analysis of head-kidney leukocytes stained with anti-CD4-2-mAb. Negative control; shaded area, Anti-CD4-2-mAb treated; black line. Immunofluorescence staining of head-kidney leukocytes incubated with anti-CD4-2 mAb (3C8), followed by FITC-conjugated goat anti-mouse IgG. Immuno-reactivity appears green.

    Techniques Used: Recombinant, Transfection, Stable Transfection, Expressing, Western Blot, Flow Cytometry, Staining, Negative Control, Immunofluorescence, Incubation

    6) Product Images from "Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein *"

    Article Title: Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.142026

    Binding of CRP to six different immobilized proteins as a function of pH and temperature. Results of a protein ligand-binding assay are shown. Microtiter wells were coated with factor H, oxLDL, C3b, IgG, Aβ, and BSA. The unreacted sites in the wells were blocked with gelatin. CRP (10 μg/ml), diluted in TBS-Ca, pH 7.0–4.6, was then added to the wells. One set of wells was incubated at 25 °C and another at 37 °C, for 2 h. Bound CRP was detected by using a rabbit polyclonal anti-CRP antibody and HRP-conjugated donkey anti-rabbit IgG. The absorbance of the developed color was read at 405 nm and plotted on the y axis.
    Figure Legend Snippet: Binding of CRP to six different immobilized proteins as a function of pH and temperature. Results of a protein ligand-binding assay are shown. Microtiter wells were coated with factor H, oxLDL, C3b, IgG, Aβ, and BSA. The unreacted sites in the wells were blocked with gelatin. CRP (10 μg/ml), diluted in TBS-Ca, pH 7.0–4.6, was then added to the wells. One set of wells was incubated at 25 °C and another at 37 °C, for 2 h. Bound CRP was detected by using a rabbit polyclonal anti-CRP antibody and HRP-conjugated donkey anti-rabbit IgG. The absorbance of the developed color was read at 405 nm and plotted on the y axis.

    Techniques Used: Binding Assay, Ligand Binding Assay, Incubation

    Structural change in CRP after binding to immobilized proteins at acidic pH. Microtiter wells were coated with factor H, oxLDL, C3b, IgG, Aβ, and BSA. CRP (10 μg/ml), diluted in TBS-Ca, pH 7.0–4.6, was then added to the wells and incubated at 37 °C for 2 h. Bound CRP was detected using three different anti-CRP antibodies: a polyclonal anti-CRP antibody, anti-CRP mAb HD2.4, or anti-CRP mAb 3H12. After addition of appropriate HRP-conjugated secondary antibodies, the absorbance of the developed color was read at 405 nm and plotted on the y axis.
    Figure Legend Snippet: Structural change in CRP after binding to immobilized proteins at acidic pH. Microtiter wells were coated with factor H, oxLDL, C3b, IgG, Aβ, and BSA. CRP (10 μg/ml), diluted in TBS-Ca, pH 7.0–4.6, was then added to the wells and incubated at 37 °C for 2 h. Bound CRP was detected using three different anti-CRP antibodies: a polyclonal anti-CRP antibody, anti-CRP mAb HD2.4, or anti-CRP mAb 3H12. After addition of appropriate HRP-conjugated secondary antibodies, the absorbance of the developed color was read at 405 nm and plotted on the y axis.

    Techniques Used: Binding Assay, Incubation

    7) Product Images from "The preclinical evaluation of immunocontraceptive vaccines based on canine zona pellucida 3 (cZP3) in a mouse model"

    Article Title: The preclinical evaluation of immunocontraceptive vaccines based on canine zona pellucida 3 (cZP3) in a mouse model

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/s12958-018-0362-x

    Antibody levels of ZP3 and GnRH in serum. Sera were collected from the mice on day 0, 14, 35, 56 and 168 after the 1st immunization. a. Ab levels of ZP3. All the serum samples were diluted (1:1000) with PBST containing 5% skim milk. HRP-conjugated goat anti-mouse IgG Ab (1:1000) was used as the second Ab. b. Ab levels of GnRH. Serum samples were collected from the mice on day 14, 56 and 168 at 1:100 dilution. Data are shown as mean ± SEM. *** P
    Figure Legend Snippet: Antibody levels of ZP3 and GnRH in serum. Sera were collected from the mice on day 0, 14, 35, 56 and 168 after the 1st immunization. a. Ab levels of ZP3. All the serum samples were diluted (1:1000) with PBST containing 5% skim milk. HRP-conjugated goat anti-mouse IgG Ab (1:1000) was used as the second Ab. b. Ab levels of GnRH. Serum samples were collected from the mice on day 14, 56 and 168 at 1:100 dilution. Data are shown as mean ± SEM. *** P

    Techniques Used: Mouse Assay

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    Incubation:

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    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli
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    Article Title: Large Extracellular Loop of Tetraspanin as a Potential Vaccine Candidate for Filariasis
    Article Snippet: Briefly, nitrocellulose membrane blotted with the each of the recombinant proteins was probed with sera collected from mice immunized with rBm TSP LEL or rWb TSP LEL for 1hr at room temperature (RT). .. After washing with PBST, membranes were incubated for 1hr at room temperature with a secondary goat anti-mouse IgG antibodies conjugated to HRP (1:5000, Thermo Fisher). .. Color was developed using diamino benzidine (Thermo Fisher Scientific) substrate.

    Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71
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    Labeling:

    Article Title: High-throughput method for in process monitoring of 3-O-sulfotransferase catalyzed sulfonation in bioengineered heparin synthesis
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    Article Title: SLC41A1 Is a Novel Mammalian Mg2+ Carrier *
    Article Snippet: His-tagged hSLC41A1 was immunoprecipitated from the membrane protein fraction with a His6 tag antibody (GenWay Biotech, San Diego, CA). .. Protein samples were separated by SDS-PAGE utilizing 12.5% polyacrylamide gels, blotted, and labeled with His6 tag antibody and goat anti-mouse (GAM)-HRP (Molecular Probes, Eugene, OR) or GAM-κ-HRP (SBA, Birmingham, AL) antibodies. .. Antibody binding was visualized using the Chemilmager™ 5500 (Alpha Innotech) or AGFA Cronex 5 medical x-ray films developed with the Curix 60 (AGFA).

    Blocking Assay:

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli
    Article Snippet: The membranes were washed and then incubated for 1 h in blocking buffer with rabbit polyclonal anti-Pet antibodies (1:500 dilution) or mouse polyclonal anti-β-barrel-translocation-unit antibodies (1:500 dilution). .. Following another wash step, the membranes were incubated for 1 h in blocking buffer with an HRP-conjugated goat anti-rabbit IgG antibody or an HRP-conjugated goat anti-mouse IgG antibody (Zymed) as indicated by the manufacturer. .. HRP was detected with the ECL reagent from Amersham.

    SDS Page:

    Article Title: SLC41A1 Is a Novel Mammalian Mg2+ Carrier *
    Article Snippet: His-tagged hSLC41A1 was immunoprecipitated from the membrane protein fraction with a His6 tag antibody (GenWay Biotech, San Diego, CA). .. Protein samples were separated by SDS-PAGE utilizing 12.5% polyacrylamide gels, blotted, and labeled with His6 tag antibody and goat anti-mouse (GAM)-HRP (Molecular Probes, Eugene, OR) or GAM-κ-HRP (SBA, Birmingham, AL) antibodies. .. Antibody binding was visualized using the Chemilmager™ 5500 (Alpha Innotech) or AGFA Cronex 5 medical x-ray films developed with the Curix 60 (AGFA).

    Article Title: Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies
    Article Snippet: SDS-PAGE and Western blottingExpression and purification processes of the soluble periplasmic scFv were visualized by SDS-PAGE and immunoblotting. .. The samples were prepared in a 5X SDS-sample buffer and boiled for 5 min at 95 °C and then was separated on two 12% SDS-PAGE at 200 V. The one gel was stained using Coomassie Brilliant Blue G-250 and the other one used for blotting on nitrocellulose membrane and immunostaining with anti-c-myc antibody 9E10 (sc-40, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a dilution of 1:1000 and 1:3000, respectively. .. Statistical analysis The statistically significant effects of each variable and the related interactions on the recovery yield of the functional scFv were confirmed by the Student’s F-test using analysis of variance (ANOVA), and a p-value less than 0.05 (p< 0.05) was statistically significant.

    Staining:

    Article Title: Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies
    Article Snippet: SDS-PAGE and Western blottingExpression and purification processes of the soluble periplasmic scFv were visualized by SDS-PAGE and immunoblotting. .. The samples were prepared in a 5X SDS-sample buffer and boiled for 5 min at 95 °C and then was separated on two 12% SDS-PAGE at 200 V. The one gel was stained using Coomassie Brilliant Blue G-250 and the other one used for blotting on nitrocellulose membrane and immunostaining with anti-c-myc antibody 9E10 (sc-40, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a dilution of 1:1000 and 1:3000, respectively. .. Statistical analysis The statistically significant effects of each variable and the related interactions on the recovery yield of the functional scFv were confirmed by the Student’s F-test using analysis of variance (ANOVA), and a p-value less than 0.05 (p< 0.05) was statistically significant.

    Immunostaining:

    Article Title: Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies
    Article Snippet: SDS-PAGE and Western blottingExpression and purification processes of the soluble periplasmic scFv were visualized by SDS-PAGE and immunoblotting. .. The samples were prepared in a 5X SDS-sample buffer and boiled for 5 min at 95 °C and then was separated on two 12% SDS-PAGE at 200 V. The one gel was stained using Coomassie Brilliant Blue G-250 and the other one used for blotting on nitrocellulose membrane and immunostaining with anti-c-myc antibody 9E10 (sc-40, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a dilution of 1:1000 and 1:3000, respectively. .. Statistical analysis The statistically significant effects of each variable and the related interactions on the recovery yield of the functional scFv were confirmed by the Student’s F-test using analysis of variance (ANOVA), and a p-value less than 0.05 (p< 0.05) was statistically significant.

    other:

    Article Title: Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein *
    Article Snippet: HRP-conjugated donkey anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG, diluted in TBS-Ca, were used (1 h at 37 °C) as the secondary antibodies.

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  • 97
    Thermo Fisher hrp conjugated goat anti mouse igg antibody
    Differential interactions between E. coli periplasmic proteins and either the Pet passenger domain or the Pet β-barrel translocation unit. Overlay assays of Pet (A) or the β-barrel translocation unit of Pet (C) and periplasmic proteins are shown. The periplasmic proteins of E. coli HB101 and UT5600 were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with purified PetS260I (A) or the β-barrel translocation unit of Pet (C). To detect specific interactions between Pet or the β-barrel translocation unit of Pet and periplasmic proteins, a primary rabbit anti-Pet antibody or a primary mouse anti-translocation-unit antibody was used, followed by an <t>HRP-conjugated</t> anti-rabbit <t>IgG</t> secondary antibody or an HRP-conjugated anti-mouse IgG secondary antibody, respectively. (B and D) Western blot assay to confirm the specific interaction between periplasmic protein and either Pet or the β-barrel translocation unit. The periplasmic fractions of E. coli , as indicated in panels A and C, were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The nonspecific interaction between the periplasmic proteins and the primary antibodies was visualized by using a rabbit anti-Pet or a mouse anti-translocation-unit primary antibody, followed by an HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody, respectively. The arrows in panel A indicate the specific interaction between Pet and two periplasmic proteins with apparent molecular masses of 37 and 50 kDa, and the asterisk in panel C indicates the specific interaction between the β-barrel translocation unit and one periplasmic protein with an apparent molecular mass of 20 kDa.
    Hrp Conjugated Goat Anti Mouse Igg Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher peroxidase conjugated goat anti mouse igg
    Effect of carrier preimmunization. Mice were preimmunized with 100 μg of each carrier in aluminum hydroxide (alum; striped bar) or with PBS in aluminum hydroxide (black bar) and then immunized twice with 0.1 μg of equivalent G1′ conjugated to rP40 or TT in the presence of aluminum hydroxide. On day 67, sera were collected and antipeptide <t>IgG</t> antibody content was measured by ELISA. Results are expressed as means ± ( n = 5).
    Peroxidase Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher goat anti human hrp conjugated igg
    Humanized anti-FSHβ antibodies specifically bind human FSH. Surface plasmon resonance (SPR, Biacore 8K/T200) was utilized to study the binding properties of human and mouse FSH with the purified humanized antibodies Hu6, Hu26, Hu28, and Hu23, as well as human–mouse chimeric molecule CH1. ( A ) Sensograms showing the concentration-dependent binding of humanized antibodies Hu6, Hu26, and Hu28 (10 to 320 nM) to human FSH. ( B ) SPR yielded measures of association constant (K a ), dissociation constant (K d ) and affinity (K D ). The K D values were consistent with in silico global net electrostatic binding energies (DDG) calculated from molecular dynamics using APBS. Hu6 displayed the highest K D and lowest K d . ( C ) ELISA showing binding of Hu6, Hu6-Fab, Hu26, Hu28, or CH1 to pituitary-derived human FSH and its recombinant glycoforms, FSH 21/18 and FSH 24 , but not to human LH, human TSH (coating at 50 ng), or Hu6-Fc detected with <t>HRP-conjugated</t> antibody to human <t>IgG.</t> There is no binding with human IgG. Data are a mean of duplicate wells. ( D ) cAMP production in differentiated 3T3.L1 cells incubated with FSH 21/18 or FSH 24 in the presence of the β 3 agonist CL316,243 ( n = 3 biological replicates). ( E ) Protein thermal shift assay utilizes Sypro-Orange to capture hydrophobic domains during heat-induced protein unfolding to yield a melting temperature (Tm). Change in fluorescence is shown as relative fluorescence units (ΔRFU) per unit change in temperature (ΔT). Hu6 showed two peaks: one at ∼69 °C, predicted to be caused by unfolding of the Fc domain, and the other at 77 °C, likely due to unfolding of Fab region. The addition of human FSH, but not human LH, produced a thermal shift solely in the putative Fab peak. Binding of FSH to the Fab domain was confirmed by a comparable thermal shift in Hu6-Fab, but not Hu6-Fc, in the presence of FSH.
    Goat Anti Human Hrp Conjugated Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher horseradish peroxidase hrp conjugated anti mouse immunoglobulin g igg
    Comparison of the binding affinities for α-DG between Old and New World arenaviruses. α-DG purified from rabbit skeletal muscle was separated by SDS-PAGE and blotted to nitrocellulose. The Old World arenaviruses LCMV WE54, LCMV ARM53b, and Lassa virus (LFV) (A) were applied at 10 7 PFU/ml and detected by the monoclonal anti-LCMV GP2 antibodies 33.6 and 86.6, using an <t>HRP-conjugated</t> anti-mouse <t>IgG</t> secondary antibody and ECL substrate. The New World arenaviruses Oliveros, Latino, Amapari, and Parana (B), each shown in a duplicate lane and used at a concentration of 10 7 PFU/ml, were detected by a 1:1,000 dilution of mouse hyperimmune serum against New World arenaviruses and an HRP-conjugated secondary antibody as for panel A.
    Horseradish Peroxidase Hrp Conjugated Anti Mouse Immunoglobulin G Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Differential interactions between E. coli periplasmic proteins and either the Pet passenger domain or the Pet β-barrel translocation unit. Overlay assays of Pet (A) or the β-barrel translocation unit of Pet (C) and periplasmic proteins are shown. The periplasmic proteins of E. coli HB101 and UT5600 were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with purified PetS260I (A) or the β-barrel translocation unit of Pet (C). To detect specific interactions between Pet or the β-barrel translocation unit of Pet and periplasmic proteins, a primary rabbit anti-Pet antibody or a primary mouse anti-translocation-unit antibody was used, followed by an HRP-conjugated anti-rabbit IgG secondary antibody or an HRP-conjugated anti-mouse IgG secondary antibody, respectively. (B and D) Western blot assay to confirm the specific interaction between periplasmic protein and either Pet or the β-barrel translocation unit. The periplasmic fractions of E. coli , as indicated in panels A and C, were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The nonspecific interaction between the periplasmic proteins and the primary antibodies was visualized by using a rabbit anti-Pet or a mouse anti-translocation-unit primary antibody, followed by an HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody, respectively. The arrows in panel A indicate the specific interaction between Pet and two periplasmic proteins with apparent molecular masses of 37 and 50 kDa, and the asterisk in panel C indicates the specific interaction between the β-barrel translocation unit and one periplasmic protein with an apparent molecular mass of 20 kDa.

    Journal: Infection and Immunity

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

    doi: 10.1128/IAI.00167-12

    Figure Lengend Snippet: Differential interactions between E. coli periplasmic proteins and either the Pet passenger domain or the Pet β-barrel translocation unit. Overlay assays of Pet (A) or the β-barrel translocation unit of Pet (C) and periplasmic proteins are shown. The periplasmic proteins of E. coli HB101 and UT5600 were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with purified PetS260I (A) or the β-barrel translocation unit of Pet (C). To detect specific interactions between Pet or the β-barrel translocation unit of Pet and periplasmic proteins, a primary rabbit anti-Pet antibody or a primary mouse anti-translocation-unit antibody was used, followed by an HRP-conjugated anti-rabbit IgG secondary antibody or an HRP-conjugated anti-mouse IgG secondary antibody, respectively. (B and D) Western blot assay to confirm the specific interaction between periplasmic protein and either Pet or the β-barrel translocation unit. The periplasmic fractions of E. coli , as indicated in panels A and C, were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The nonspecific interaction between the periplasmic proteins and the primary antibodies was visualized by using a rabbit anti-Pet or a mouse anti-translocation-unit primary antibody, followed by an HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody, respectively. The arrows in panel A indicate the specific interaction between Pet and two periplasmic proteins with apparent molecular masses of 37 and 50 kDa, and the asterisk in panel C indicates the specific interaction between the β-barrel translocation unit and one periplasmic protein with an apparent molecular mass of 20 kDa.

    Article Snippet: Following another wash step, the membranes were incubated for 1 h in blocking buffer with an HRP-conjugated goat anti-rabbit IgG antibody or an HRP-conjugated goat anti-mouse IgG antibody (Zymed) as indicated by the manufacturer.

    Techniques: Positron Emission Tomography, Translocation Assay, SDS Page, Incubation, Purification, Western Blot

    Affinity of Pet for purified VirK. (A) E. coli BL21 was fractionated as described in Materials and Methods. Cytoplasmic, IM, periplasmic, and OM fractions were analyzed by Western blot (WB) assay using anti-His antibodies and, as markers, antibodies against GroEL and β-lactamase. (B) Bacterial cultures transformed with pVirK-His were lysed 4 h after IPTG induction. VirK-His 6 was purified from cell lysates supernatant by Ni-NTA agarose affinity chromatography in accordance with the manufacturer's instructions (Qiagen). Fractions from different stages of the purification process were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. VirK-His 6 was visualized using a mouse anti-His primary antibody and an HRP-conjugated anti-mouse IgG secondary antibody. The arrow indicates the recombinant VirK protein. (C and D) Purified Pet, purified and denatured Pet, the purified β-domain, or the purified and denatured β-domain was incubated with VirK-His. Parallel samples were incubated with fractions 2 and 3 from BL21 transformed with the empty vector (pRSETA) as a negative control. After an overnight incubation, the complexes were immunoprecipitated with anti-His antibodies. As an additional control, the antibodies were incubated in the absence of either Pet or the β domain. The immunocomplexes (C) and nonimmunoprecipitated proteins (D) were separated by SDS-PAGE and visualized with Coomassie brilliant blue stain. MWM, MW markers.

    Journal: Infection and Immunity

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

    doi: 10.1128/IAI.00167-12

    Figure Lengend Snippet: Affinity of Pet for purified VirK. (A) E. coli BL21 was fractionated as described in Materials and Methods. Cytoplasmic, IM, periplasmic, and OM fractions were analyzed by Western blot (WB) assay using anti-His antibodies and, as markers, antibodies against GroEL and β-lactamase. (B) Bacterial cultures transformed with pVirK-His were lysed 4 h after IPTG induction. VirK-His 6 was purified from cell lysates supernatant by Ni-NTA agarose affinity chromatography in accordance with the manufacturer's instructions (Qiagen). Fractions from different stages of the purification process were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. VirK-His 6 was visualized using a mouse anti-His primary antibody and an HRP-conjugated anti-mouse IgG secondary antibody. The arrow indicates the recombinant VirK protein. (C and D) Purified Pet, purified and denatured Pet, the purified β-domain, or the purified and denatured β-domain was incubated with VirK-His. Parallel samples were incubated with fractions 2 and 3 from BL21 transformed with the empty vector (pRSETA) as a negative control. After an overnight incubation, the complexes were immunoprecipitated with anti-His antibodies. As an additional control, the antibodies were incubated in the absence of either Pet or the β domain. The immunocomplexes (C) and nonimmunoprecipitated proteins (D) were separated by SDS-PAGE and visualized with Coomassie brilliant blue stain. MWM, MW markers.

    Article Snippet: Following another wash step, the membranes were incubated for 1 h in blocking buffer with an HRP-conjugated goat anti-rabbit IgG antibody or an HRP-conjugated goat anti-mouse IgG antibody (Zymed) as indicated by the manufacturer.

    Techniques: Positron Emission Tomography, Purification, Western Blot, Transformation Assay, Affinity Chromatography, SDS Page, Recombinant, Incubation, Plasmid Preparation, Negative Control, Immunoprecipitation, Staining

    Secretion and OM insertion of Pet by E. coli MC4100Δ skp . (A) Supernatants were collected from overnight cultures of untransformed E. coli strains that did not express Pet (HB101 and MC4100Δ skp ) or from the same E. coli strains transformed with a plasmid encoding wild-type Pet (pCEFN1). Proteins in the supernatants were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The secreted pool of Pet was detected by Western blot analysis using a rabbit anti-Pet polyclonal primary antibody and an HRP-conjugated anti-rabbit IgG secondary antibody. Supernatants were obtained from bacterial cultures (optical density at 600 nm of 1.0) under the same conditions for each strain and then precipitated by using 20% TCA. (B) OM proteins from the strains used in panel A were separated by SDS-PAGE and visualized by Coomassie blue staining. Arrows denote the β-barrel translocation unit of Pet which was embedded in the OM. MWM, MW markers.

    Journal: Infection and Immunity

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

    doi: 10.1128/IAI.00167-12

    Figure Lengend Snippet: Secretion and OM insertion of Pet by E. coli MC4100Δ skp . (A) Supernatants were collected from overnight cultures of untransformed E. coli strains that did not express Pet (HB101 and MC4100Δ skp ) or from the same E. coli strains transformed with a plasmid encoding wild-type Pet (pCEFN1). Proteins in the supernatants were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The secreted pool of Pet was detected by Western blot analysis using a rabbit anti-Pet polyclonal primary antibody and an HRP-conjugated anti-rabbit IgG secondary antibody. Supernatants were obtained from bacterial cultures (optical density at 600 nm of 1.0) under the same conditions for each strain and then precipitated by using 20% TCA. (B) OM proteins from the strains used in panel A were separated by SDS-PAGE and visualized by Coomassie blue staining. Arrows denote the β-barrel translocation unit of Pet which was embedded in the OM. MWM, MW markers.

    Article Snippet: Following another wash step, the membranes were incubated for 1 h in blocking buffer with an HRP-conjugated goat anti-rabbit IgG antibody or an HRP-conjugated goat anti-mouse IgG antibody (Zymed) as indicated by the manufacturer.

    Techniques: Positron Emission Tomography, Transformation Assay, Plasmid Preparation, SDS Page, Western Blot, Staining, Translocation Assay

    Inhibition of Pet AT but not porin secretion from virK null mutants. (A) Supernatants were collected from overnight cultures of an E. coli laboratory strain that does not express Pet (HB101), a pathogenic E. coli strain that expresses Pet (EAEC), isogenic strain EAECΔ virK , and virK -complemented strain EAECΔ virK /pVirK. Proteins in the supernatants were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The secreted pools of Pet were detected by Western blot (WB) analysis using a rabbit anti-Pet primary antibody, followed by an HRP-conjugated anti-rabbit IgG secondary antibody. (B) OMPs were obtained from cultures of EAEC, EAECΔ virK , and EAECΔ virK /pVirK; the purified β domain was used as a control. The OMPs were separated by SDS-PAGE and visualized with Coomassie brilliant blue stain. Arrows indicate the porins and the β-barrel translocation unit. For all experiments, supernatants from the bacterial strains were normalized by growth under equal conditions and sample harvesting at the same growth phase (optical density at 600 nm of 1.0) before precipitation with 20% TCA. (C) Retention and degradation of Pet AT protein in a virK mutant strain. Periplasmic fractions were obtained from overnight cultures of EAEC and its isogenic virK mutant. Samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The periplasmic pool of Pet was detected by Western blot analysis using a rabbit anti-Pet primary antibody, followed by an HRP-conjugated anti-rabbit IgG secondary antibody.

    Journal: Infection and Immunity

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

    doi: 10.1128/IAI.00167-12

    Figure Lengend Snippet: Inhibition of Pet AT but not porin secretion from virK null mutants. (A) Supernatants were collected from overnight cultures of an E. coli laboratory strain that does not express Pet (HB101), a pathogenic E. coli strain that expresses Pet (EAEC), isogenic strain EAECΔ virK , and virK -complemented strain EAECΔ virK /pVirK. Proteins in the supernatants were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The secreted pools of Pet were detected by Western blot (WB) analysis using a rabbit anti-Pet primary antibody, followed by an HRP-conjugated anti-rabbit IgG secondary antibody. (B) OMPs were obtained from cultures of EAEC, EAECΔ virK , and EAECΔ virK /pVirK; the purified β domain was used as a control. The OMPs were separated by SDS-PAGE and visualized with Coomassie brilliant blue stain. Arrows indicate the porins and the β-barrel translocation unit. For all experiments, supernatants from the bacterial strains were normalized by growth under equal conditions and sample harvesting at the same growth phase (optical density at 600 nm of 1.0) before precipitation with 20% TCA. (C) Retention and degradation of Pet AT protein in a virK mutant strain. Periplasmic fractions were obtained from overnight cultures of EAEC and its isogenic virK mutant. Samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The periplasmic pool of Pet was detected by Western blot analysis using a rabbit anti-Pet primary antibody, followed by an HRP-conjugated anti-rabbit IgG secondary antibody.

    Article Snippet: Following another wash step, the membranes were incubated for 1 h in blocking buffer with an HRP-conjugated goat anti-rabbit IgG antibody or an HRP-conjugated goat anti-mouse IgG antibody (Zymed) as indicated by the manufacturer.

    Techniques: Inhibition, Positron Emission Tomography, SDS Page, Western Blot, Purification, Staining, Translocation Assay, Mutagenesis

    Effect of carrier preimmunization. Mice were preimmunized with 100 μg of each carrier in aluminum hydroxide (alum; striped bar) or with PBS in aluminum hydroxide (black bar) and then immunized twice with 0.1 μg of equivalent G1′ conjugated to rP40 or TT in the presence of aluminum hydroxide. On day 67, sera were collected and antipeptide IgG antibody content was measured by ELISA. Results are expressed as means ± ( n = 5).

    Journal: Infection and Immunity

    Article Title: Carrier Properties of a Protein Derived from Outer Membrane Protein A of Klebsiella pneumoniae

    doi:

    Figure Lengend Snippet: Effect of carrier preimmunization. Mice were preimmunized with 100 μg of each carrier in aluminum hydroxide (alum; striped bar) or with PBS in aluminum hydroxide (black bar) and then immunized twice with 0.1 μg of equivalent G1′ conjugated to rP40 or TT in the presence of aluminum hydroxide. On day 67, sera were collected and antipeptide IgG antibody content was measured by ELISA. Results are expressed as means ± ( n = 5).

    Article Snippet: Peroxidase-conjugated goat anti-mouse IgG (Pierce) or goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates, Birmingham, Ala.) was reacted with each well for 1 h at 37°C.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Antipolysaccharide antibody responses. Mice were immunized three times with 10 μg of Hib polysaccharide conjugated to rP40 or TT in presence of aluminum hydroxide. After one (white bars), two (horizontal bars), or three (black bars) immunizations, sera were collected and polysaccharide-specific IgG antibody content was measured by ELISA. Results are expressed as means ± SD ( n = 5).

    Journal: Infection and Immunity

    Article Title: Carrier Properties of a Protein Derived from Outer Membrane Protein A of Klebsiella pneumoniae

    doi:

    Figure Lengend Snippet: Antipolysaccharide antibody responses. Mice were immunized three times with 10 μg of Hib polysaccharide conjugated to rP40 or TT in presence of aluminum hydroxide. After one (white bars), two (horizontal bars), or three (black bars) immunizations, sera were collected and polysaccharide-specific IgG antibody content was measured by ELISA. Results are expressed as means ± SD ( n = 5).

    Article Snippet: Peroxidase-conjugated goat anti-mouse IgG (Pierce) or goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates, Birmingham, Ala.) was reacted with each well for 1 h at 37°C.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Humanized anti-FSHβ antibodies specifically bind human FSH. Surface plasmon resonance (SPR, Biacore 8K/T200) was utilized to study the binding properties of human and mouse FSH with the purified humanized antibodies Hu6, Hu26, Hu28, and Hu23, as well as human–mouse chimeric molecule CH1. ( A ) Sensograms showing the concentration-dependent binding of humanized antibodies Hu6, Hu26, and Hu28 (10 to 320 nM) to human FSH. ( B ) SPR yielded measures of association constant (K a ), dissociation constant (K d ) and affinity (K D ). The K D values were consistent with in silico global net electrostatic binding energies (DDG) calculated from molecular dynamics using APBS. Hu6 displayed the highest K D and lowest K d . ( C ) ELISA showing binding of Hu6, Hu6-Fab, Hu26, Hu28, or CH1 to pituitary-derived human FSH and its recombinant glycoforms, FSH 21/18 and FSH 24 , but not to human LH, human TSH (coating at 50 ng), or Hu6-Fc detected with HRP-conjugated antibody to human IgG. There is no binding with human IgG. Data are a mean of duplicate wells. ( D ) cAMP production in differentiated 3T3.L1 cells incubated with FSH 21/18 or FSH 24 in the presence of the β 3 agonist CL316,243 ( n = 3 biological replicates). ( E ) Protein thermal shift assay utilizes Sypro-Orange to capture hydrophobic domains during heat-induced protein unfolding to yield a melting temperature (Tm). Change in fluorescence is shown as relative fluorescence units (ΔRFU) per unit change in temperature (ΔT). Hu6 showed two peaks: one at ∼69 °C, predicted to be caused by unfolding of the Fc domain, and the other at 77 °C, likely due to unfolding of Fab region. The addition of human FSH, but not human LH, produced a thermal shift solely in the putative Fab peak. Binding of FSH to the Fab domain was confirmed by a comparable thermal shift in Hu6-Fab, but not Hu6-Fc, in the presence of FSH.

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

    Article Title: First-in-class humanized FSH blocking antibody targets bone and fat

    doi: 10.1073/pnas.2014588117

    Figure Lengend Snippet: Humanized anti-FSHβ antibodies specifically bind human FSH. Surface plasmon resonance (SPR, Biacore 8K/T200) was utilized to study the binding properties of human and mouse FSH with the purified humanized antibodies Hu6, Hu26, Hu28, and Hu23, as well as human–mouse chimeric molecule CH1. ( A ) Sensograms showing the concentration-dependent binding of humanized antibodies Hu6, Hu26, and Hu28 (10 to 320 nM) to human FSH. ( B ) SPR yielded measures of association constant (K a ), dissociation constant (K d ) and affinity (K D ). The K D values were consistent with in silico global net electrostatic binding energies (DDG) calculated from molecular dynamics using APBS. Hu6 displayed the highest K D and lowest K d . ( C ) ELISA showing binding of Hu6, Hu6-Fab, Hu26, Hu28, or CH1 to pituitary-derived human FSH and its recombinant glycoforms, FSH 21/18 and FSH 24 , but not to human LH, human TSH (coating at 50 ng), or Hu6-Fc detected with HRP-conjugated antibody to human IgG. There is no binding with human IgG. Data are a mean of duplicate wells. ( D ) cAMP production in differentiated 3T3.L1 cells incubated with FSH 21/18 or FSH 24 in the presence of the β 3 agonist CL316,243 ( n = 3 biological replicates). ( E ) Protein thermal shift assay utilizes Sypro-Orange to capture hydrophobic domains during heat-induced protein unfolding to yield a melting temperature (Tm). Change in fluorescence is shown as relative fluorescence units (ΔRFU) per unit change in temperature (ΔT). Hu6 showed two peaks: one at ∼69 °C, predicted to be caused by unfolding of the Fc domain, and the other at 77 °C, likely due to unfolding of Fab region. The addition of human FSH, but not human LH, produced a thermal shift solely in the putative Fab peak. Binding of FSH to the Fab domain was confirmed by a comparable thermal shift in Hu6-Fab, but not Hu6-Fc, in the presence of FSH.

    Article Snippet: Serum Hu6 levels were measured by an in-house ELISA, in which plates were coated with goat anti-human IgG Fab antibody (200 µg per well; Invitrogen, no. 31122) and incubated with mouse serum (1:1,000) followed by capture with goat anti-human HRP-conjugated IgG (H + L) antibody (Invitrogen, A18805).

    Techniques: SPR Assay, Binding Assay, Purification, Concentration Assay, In Silico, Enzyme-linked Immunosorbent Assay, Derivative Assay, Recombinant, Incubation, Thermal Shift Assay, Fluorescence, Produced

    Comparison of the binding affinities for α-DG between Old and New World arenaviruses. α-DG purified from rabbit skeletal muscle was separated by SDS-PAGE and blotted to nitrocellulose. The Old World arenaviruses LCMV WE54, LCMV ARM53b, and Lassa virus (LFV) (A) were applied at 10 7 PFU/ml and detected by the monoclonal anti-LCMV GP2 antibodies 33.6 and 86.6, using an HRP-conjugated anti-mouse IgG secondary antibody and ECL substrate. The New World arenaviruses Oliveros, Latino, Amapari, and Parana (B), each shown in a duplicate lane and used at a concentration of 10 7 PFU/ml, were detected by a 1:1,000 dilution of mouse hyperimmune serum against New World arenaviruses and an HRP-conjugated secondary antibody as for panel A.

    Journal: Journal of Virology

    Article Title: New World Arenavirus Clade C, but Not Clade A and B Viruses, Utilizes ?-Dystroglycan as Its Major Receptor

    doi: 10.1128/JVI.76.10.5140-5146.2002

    Figure Lengend Snippet: Comparison of the binding affinities for α-DG between Old and New World arenaviruses. α-DG purified from rabbit skeletal muscle was separated by SDS-PAGE and blotted to nitrocellulose. The Old World arenaviruses LCMV WE54, LCMV ARM53b, and Lassa virus (LFV) (A) were applied at 10 7 PFU/ml and detected by the monoclonal anti-LCMV GP2 antibodies 33.6 and 86.6, using an HRP-conjugated anti-mouse IgG secondary antibody and ECL substrate. The New World arenaviruses Oliveros, Latino, Amapari, and Parana (B), each shown in a duplicate lane and used at a concentration of 10 7 PFU/ml, were detected by a 1:1,000 dilution of mouse hyperimmune serum against New World arenaviruses and an HRP-conjugated secondary antibody as for panel A.

    Article Snippet: Horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) was from Pierce Chemical Co. (Rockford, Ill.).

    Techniques: Binding Assay, Purification, SDS Page, Concentration Assay