Total protein extracts from plant samples were prepared and resolved by SDS-PAGE, as described (Pasin et al. 2020 (link)). Immuno-detection was conducted using a rabbit anti-TVMV coat protein serum (Domier et al. 1989 (link)) as the primary antibody, and a peroxidase goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) as the conjugate. Virion micrographs were obtained by immunosorbent electron microscopy as described (Valli et al. 2014 (link)). Briefly, plant extracts were prepared in 5 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5 mM DTT, and incubated with collodion-coated carbon-stabilized copper grids precoated with the anti-TVMV CP serum. Grids were negative-stained with 2% uranyl acetate and observed in a transmission electron microscope (JEM 1011, Jeol); images were taken with the ES1000W Erlangshen CCD camera (Gatan). ImageJ (Schneider et al. 2012 (link)) was used for image processing.
Peroxidase goat anti rabbit igg
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom
Peroxidase goat anti-rabbit IgG is a secondary antibody conjugate that binds to rabbit immunoglobulin G (IgG) molecules. It is labeled with the enzyme peroxidase, which can be used to detect and visualize the presence of rabbit IgG in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Fl 335, by Santa Cruz Biotechnology (3 mentions)
Jem 1011, by JEOL (2 mentions)
Es1000w erlangshen ccd camera, by Ametek (2 mentions)
Peroxidase goat anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Vectastain abc kit, by Vector Laboratories (2 mentions)
Ab3594, by Abcam (2 mentions)
Ab64077, by Abcam (2 mentions)
Sc 2027, by Santa Cruz Biotechnology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Anti c met, by Santa Cruz Biotechnology (2 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Pierce ecl, by Thermo Fisher Scientific (1 mentions)
Axiocam mrc5, by Zeiss (1 mentions)
Axio imager a1 microscope, by Zeiss (1 mentions)
12 protocols using peroxidase goat anti rabbit igg
1
Immunodetection and Visualization of TVMV
2
Western Blot and Immunofluorescence Analysis of IL-10RA
3
Immunohistochemical Analysis of Epigenetic Regulators
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Immunohistochemistry was performed on 5 μm thick paraffin-embedded EDTA-decalcified knee sections. Heat induced epitope retrieval was performed using a Citrate-EDTA buffer (pH 6.2) for 10 minutes at 95°C. Sections were treated with 3% H2O2/methanol for 10 minutes to inactivate endogenous peroxidase, blocked in goat serum for 30 minutes, and incubated overnight at 4°C with primary antibodies against DOT1L (Abcam, ab64077, 6 μg/ml) or HIF1A (Abcam, ab82832, 10 μg/ml) or for 90 minutes with primary antibody against H3K79me2 (Abcam, ab3594, 1 μg/ml). Rabbit IgG (Santa Cruz, sc-2027) was used as negative control. Avidin-biotin complex amplification (Vectastain ABC kit, Vector Laboratories) was used, except for the immunohistochemical detection of H3K79me2. Peroxidase goat anti-rabbit IgG (Jackson Immunoresearch) was applied for 30 minutes, and peroxidase activity was determined using DAB. Images were taken using an Olympus IX83 microscope. Quantification of the DAB staining was performed with a color deconvolution plugin (Jacqui Ross, Auckland University) in ImageJ Software (NIH Image). Quantification was performed using the average of 2 technical replicates for 5 different mice per condition, with staining intensity reported relative to the average of the 5 SHAM+Vehicle mice.
4
Molecular Responses to tRNA/mir-34a and Doxorubicin in 143B Cells
5
Combinatorial Treatment Effects on c-MET and p-Erk1/2
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Human OS 143B-GFP-Luc cells were treated with doxorubicin (40 nM), sorafenib (4 μM) and miR-34a (12 nM) alone or in combination. Cells were collected at 72 h post-treatment and lysed in RIPA buffer containing protease inhibitor cocktail. Following determination of protein concentrations with a BCA Protein Assay Kit, protein samples (30 μg/lane) were loaded and separated on a 10% SDS-PAGE gel and transferred onto PVDF membranes. Membranes were incubated with anti-c-MET (1:200; C-28; Santa Cruz, Dallas, Texas), anti-p-Erk1/2 (1:1000; Thr202/Tyr204; Cell Signaling Technology), or anti-GAPDH (1:2,000; FL-335; Santa Cruz) rabbit antibody, and then with a peroxidase goat anti-rabbit IgG (Jackson ImmunoResearch Inc., West Grove, PA, USA). After incubation with Clarity Western ECL substrates (Bio-Rad, Hercules, CA, USA), the proteins were visualized with the ChemiDoc MP Imaging System (Bio-Rad) and quantified by Image Lab software (Bio-Rad). GAPDH was used as a loading control.
6
ELISPOT Quantification of Antibody Secreting Cells
7
Western Blot Analysis of Protein Targets
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Cells were seeded in six-well plates and treated with 4 nM tRNA/mir-34a, tRNA/MSA or vehicle, as described above. 72 h post-transfection, cells were harvested with trypsin and washed twice with cold PBS. Cell lysates were prepared using RIPA buffer (Rockland Immunochemical Inc., Limerick, PA) supplemented with the complete protease inhibitor cocktail (Roche, Nutley, NJ). Protein concentrations were quantitated with a BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Whole cell proteins (40 μg/lane) were separated on a 10% SDS-PAGE gel and electro-transferred onto PVDF membranes using Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were incubated with anti-SIRT1 (1:500 dilution; H-300; Santa Cruz Biotech Inc., Texas, TX), anti-c-MET (1:200; C-28; Santa Cruz), anti-BCL2 (1:200; N-19; Santa Cruz), anti-CDK6 (1:1000; C-21; Santa Cruz), or anti-GAPDH (1:2,000; FL-335; Santa Cruz) rabbit polyclonal antibody. The membranes were then blotted with a peroxidase goat anti-rabbit IgG (Jackson ImmunoResearch Inc., West Grove, PA), incubated with Clarity Western ECL substrates (Bio-Rad), and visualized with the ChemiDoc MP Imaging System (Bio-Rad).
8
Immunodetection and Visualization of TVMV
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Total protein extracts from plant samples were prepared and resolved by SDS-PAGE, as described (Pasin et al. 2020 (link)). Immuno-detection was conducted using a rabbit anti-TVMV coat protein serum (Domier et al. 1989 (link)) as the primary antibody, and a peroxidase goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) as the conjugate. Virion micrographs were obtained by immunosorbent electron microscopy as described (Valli et al. 2014 (link)). Briefly, plant extracts were prepared in 5 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5 mM DTT, and incubated with collodion-coated carbon-stabilized copper grids precoated with the anti-TVMV CP serum. Grids were negative-stained with 2% uranyl acetate and observed in a transmission electron microscope (JEM 1011, Jeol); images were taken with the ES1000W Erlangshen CCD camera (Gatan). ImageJ (Schneider et al. 2012 (link)) was used for image processing.
9
Antibody Sourcing for Protein Analysis
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Antibodies (Abs) anti-P4D1 ubiquitin, -β2, -PSMD2, -p62 were purchased from Santa Cruz Biotechnology (CA). Abs anti-α6 and -PSMD3 were obtained from Invitrogen. Anti-β-actin Ab was from Abgent. Anti-annexin 5 Ab was from BD Science. Abs anti-β1, -RPN10 and -RPT4 were from Enzo Life Science. Anti-β5, -PSMA2, -LC3B, -ADRM1, -AMBRA1 and -HYOU1 were from Cell Signaling Technology (Danvers, MA, USA). Abs anti-mTOR and -S100A9 were from Sigma (St. Louis, MO, USA). Abs anti-UVRAG, -TOLLIP, -VPS33A, and -VTI1A were from Thermo Fisher Scientific (Waltham, MA, USA). Donkey anti-rabbit TexRed and donkey anti-mouse FITC secondary Abs were from Jackson Immunoresearch. Goat anti-mouse Star 635 was from Abberior and Donkey anti-Rabbit Alexa Fluor 594 was from Invitrogen. Fluorescent secondary Abs IRDye® 800CW and IRDye® 680RD were from LICOR. Peroxidase Goat Anti-Rabbit IgG and Peroxidase Rabbit Anti-Mouse IgG were from Jackson Immunoresearch.
10
Immunofluorescence and Immunoblotting Assay
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