Chromatin immunoprecipitation (ChIP) assay was performed as described previously45 (link) with β-hydroxybutyryl-Histone H3(Lys9) rabbit pAb (PTM BIOLABS, Chicago, IL), anti-acetyl-Histone H3(Lys9) pAb (Merck Millipore, Burlington, NJ), anti-Histone H3(dimethyl K9) antibody (Abcam) ChromPure Rabbit IgG (Jackson Immuno Research, Baltimore, PA), and ChromPure Rabbit IgG (Jackson Immuno Research), and the primers described in Supplementary Table 3 .
Chrompure rabbit igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
ChromPure Rabbit IgG is a purified rabbit immunoglobulin G (IgG) product. It is a laboratory reagent intended for use in various immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Aminolink plus immobilization kit, by Thermo Fisher Scientific (2 mentions)
Ab100792, by Abcam (2 mentions)
Protein a coated beads, by Santa Cruz Biotechnology (2 mentions)
Vectastain elite abc kit for mouse igg, by Vector Laboratories (2 mentions)
Biotinylated goat anti rabbit igg ba 1000, by Vector Laboratories (2 mentions)
Protein a agarose beads, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse antibody, by Merck Group (1 mentions)
Chrompure mouse igg, by Jackson ImmunoResearch (1 mentions)
Affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Laminin, by Merck Group (1 mentions)
10 protocols using chrompure rabbit igg
1
Histone Modification Analysis by ChIP
2
Antibody Immobilization on Agarose Beads
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Rabbit anti-mouse GPR124 ECD antibody (500 μg antibody/100 μl beads), ChromPure rabbit IgG (Jackson ImmunoResearch #011-000-003, 500 μg antibody/100 μl beads), or carrier-free anti-WNT7A antibody (abcam #ab100792, 100 μg antibody/100 μl beads) were covalently coupled to agarose beads using AminoLink Plus Immobilization Kit (ThermoFisher Scientific) according to the manufacturer’s protocol.
3
Immunoprecipitation of Pannexin-1 and CRMP2
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N2a cells (4.5 × 107 cells/immunoprecipitation (IP)), or VZ tissue dissected from pooled P0–P10 or P60 C57BL/6J mice were homogenized in TBS lysis buffer (10 mM Tris base, pH 7.4, 150 mM NaCl, 1% IGEPAL), supplemented with protease inhibitor cocktail, PMSF, and sodium orthovanadate, for 30 min on ice, followed by centrifugation at 4°C for 20 min at 12,000 rpm to remove debris. The supernatant was pre-cleared for 45–60 min with protein-A agarose beads (MilliporeSigma, 11134515001 ROCHE) cross-linked to ChromPure rabbit IgG (Jackson ImmunoResearch, 011-000-003) at 4°C with shaking. Pre-cleared lysate (1.5–2.5 mg) was added to 200 μL protein-A bead suspension cross-linked with 5 μg of Panx1-EL2 antibody (generously provided by Drs. Dale Laird and Silvia Penuela, University of Western Ontario, Canada), Crmp2 polyclonal antibody (Bioss Antibodies, BS-1790R), or ChromPure rabbit IgG control, and incubated 1.5 h at 4°C with shaking. Beads were washed 2–3 times with TBS/0.5% IGEPAL and four times with TBS, then eluted in two bead volumes of 0.5 M ammonium hydroxide/0.5 mM EDTA for 30 min at room temperature with shaking. The eluent was dried and rehydrated in TBS lysis buffer with SDS-PAGE loading dye under reducing conditions to analyze by Western blotting.
4
Antibody Characterization Protocol
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Antibodies were obtained as follows—LEAF purified mouse IgG1 (401404); LEAF purified mouse IgM (401604, Biolegend, San Diego, CA, USA); mouse anti-phosphotyrosine mAb (Life Technologies, Carlsbad, CA, USA); mouse monoclonal anti-beta-actin Ab (A2228), mouse monoclonal anti-glycophorin A IgG (clone R10, Lot B0607, sc-53905); mouse monoclonal anti-GPA IgM (clone E4, G7900, Sigma-Aldrich, St Louis, MO, USA); anti-CR1 mAb 1F11 (gift of Henry Marsh, Celldex Therapeutics, Needham, MA, USA); mouse monoclonal anti-CFTR (CF3) (ab2784, both Abcam, Cambridge, MA, USA); AffiniPure goat anti-mouse IgG (115-005-003); ChromPure rabbit IgG (011-000-003); ChromPure mouse IgG (015-000-003, Jackson ImmunoResearch, West Grove, PA, USA); HRP conjugated goat anti-mouse antibody (Millipore, Billerica, MA, USA); mouse monoclonal anti-human C3d (A207), mouse monoclonal anti-human C4d (A213, Quidel, San Diego, CA, USA).
5
Depletion of α-MSH and NPY from RPE Media
6
Antibody Immobilization on Agarose Beads
7
Depletion of α-MSH and NPY from RPE Media
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Antibodies to α-MSH and NPY (Bachem Americas Inc., Torrance, CA) were added to the ARPE-19 established monolayer conditioned media. For a control ChromPure Rabbit IgG (Jackson ImmunoResearch Laboratory, West Grove, PA) was used. Antibody concentrations were 2 μg/ml, a level that would bind a molar equivalent of 1 mg of α-MSH and NPY. This far in excess of what has been measured in culture for RPE eyecups [4 (link)]. The conditioned media with antibody was incubated 1 hour at 37°C. Protein-A coated beads (Santa Cruz Biotechnology) were add and the mixture was incubated for an additional 1 hour at 37°C. The beads were centrifuged down, and the α-MSH/NPY-depleted supernatant was used in the phagolysosome assay.
8
Laminin-Mediated Transwell Migration Assay
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Pt45.P1 cells were suspended in serum free DMEM and pretreated with the indicated antibody at a concentration of 50 µg/mL for 30 minutes. 1.5 x 104 cells were then added to serum free DMEM in 8.0 µm transwells (Corning 353097) precoated with 1 µg laminin (Sigma L4544). 2% FBS in DMEM was used as the source of chemoattractants. ChromPure rabbit IgG (Jackson ImmunoResearch 011-000-003) was used as an isotype control.
9
Immunohistochemical Detection of IL-23 in Reproductive Tissues
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Transverse (oviduct) or longitudinal (uterus) cryosections of 7 µm were blocked in 5% goat serum and incubated overnight at 4°C with a rabbit anti-human IL-23 antibody (H-113; Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted to 0.2 µg/ml. Negative controls included sections incubated without primary antibody and sections incubated with ChromPure Rabbit IgG (011-000-003, Jackson ImmunoResearch Laboratories Inc., West Grove, PA). IL-23-like immunolabelling was detected using Vectastain ® Elite ABC kit for Mouse IgG (PK-6102; Vector Laboratories Inc., Burlingame, CA) with biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories Inc.) diluted 1/1000 replacing anti-mouse IgG. Endogenous peroxidase activity was blocked, 3, 3'-diaminobenzidine tablets (D-5905; Sigma-Aldrich Inc., Saint Louis, MO) were used for visualization and sections were counterstained with Mayer's Hematoxylin.
For semi-quanti cation of immunolabelling, photographs were taken with identical exposure settings.
Immunolabelling intensity of endometrium and endosalpinx was scored (blind by three persons) from 0-4, where 0 corresponds to the intensity of the negative controls and 4 to very high intensity.
For semi-quanti cation of immunolabelling, photographs were taken with identical exposure settings.
Immunolabelling intensity of endometrium and endosalpinx was scored (blind by three persons) from 0-4, where 0 corresponds to the intensity of the negative controls and 4 to very high intensity.
10
Immunohistochemical Detection of IL-23 in Reproductive Tissues
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Transverse (oviduct) or longitudinal (uterus) cryosections of 7 µm were blocked in 5% goat serum and incubated overnight at 4°C with a rabbit anti-human IL-23 antibody (H-113; Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted to 0.2 µg/ml. Negative controls included sections incubated without primary antibody and sections incubated with ChromPure Rabbit IgG (011-000-003, Jackson ImmunoResearch Laboratories Inc., West Grove, PA). IL-23-like immunolabelling was detected using Vectastain ® Elite ABC kit for Mouse IgG (PK-6102; Vector Laboratories Inc., Burlingame, CA) with biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories Inc.) diluted 1/1000 replacing anti-mouse IgG. Endogenous peroxidase activity was blocked, 3, 3'-diaminobenzidine tablets (D-5905; Sigma-Aldrich Inc., Saint Louis, MO) were used for visualization and sections were counterstained with Mayer's Hematoxylin.
For semi-quanti cation of immunolabelling, photographs were taken with identical exposure settings.
Immunolabelling intensity of endometrium and endosalpinx was scored (blind by three persons) from 0-4, where 0 corresponds to the intensity of the negative controls and 4 to very high intensity.
For semi-quanti cation of immunolabelling, photographs were taken with identical exposure settings.
Immunolabelling intensity of endometrium and endosalpinx was scored (blind by three persons) from 0-4, where 0 corresponds to the intensity of the negative controls and 4 to very high intensity.
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