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Hrp conjugated anti rabbit igg h l

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HRP-conjugated anti-rabbit IgG (H+L) is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and visualize rabbit primary antibodies in various immunoassay applications.

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8 protocols using hrp conjugated anti rabbit igg h l

1

Western Blot Analysis of STAT3 and STAT5

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Immobilon-P PVDF membranes (Millipore) were probed with the primary antibodies at 4°C overnight and then washed and incubated with HRP-conjugated goat anti-mouse IgG (H+L) or anti-rabbit IgG (H+L) secondary antibody (Promega). The following primary antibodies were used: anti-STAT3 (clone D3Z2G, Cell Signaling Technology), anti-phospho-STAT3 (Tyr705) (clone D3A7, Cell Signaling Technology), anti-STAT5 (Cell Signaling Technology), anti-phospho-STAT5 (Tyr694) (clone D47E7, Cell Signaling Technology), anti-β-actin (clone C4, Santa Cruz Biotechnology, sc-47778), HRP-conjugated anti-mouse IgG (H+L) (Promega, W4021), and HRP-conjugated anti-rabbit IgG (H+L) (Promega, W4011). All images were acquired with ChemiDoc MP system (Bio-Rad) and Image Lab software (Bio-Rad). Protein levels for each blot were quantified with ImageJ software.
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2

Immunohistochemical Staining of T Cell Subsets

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Paraffin-embedded tumor tissues were used in the immunohistochemical staining of CD4+ and CD8+ T cells. A microtome (Leica, Wetzlar, DEU) was used to prepare the 4-µm-thick tissue sections before deparaffinization and rehydration. Antigen epitopes were retrieved by autoclaving at 121 °C for 20 min in 10 mM sodium citrate buffer (pH 6.0). The sections were incubated overnight at 4 °C with the rabbit anti-CD4 (Bioss, Boston, MA) or anti-CD8 (Abcam, Cambridge, UK) antibody that was diluted 1:100. Subsequently, the sections were incubated for 60 min at room temperature with the secondary antibody HRP-conjugated anti-rabbit IgG (H + L) (Promega, Madison, WI). Positive signals were visualized by using 3,3’-diaminobenzidine (Sigma-Aldrich) for 3 min before the sections were counterstained with hematoxylin for 5 min. As negative controls, the sections were stained in parallel with isotype control, the rabbit monoclonal IgG antibody (Abcam). CD4- or CD8-positive cells were counted as described previously [11 (link)].
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3

Western Blot Analysis of Cellular Signaling

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Protein samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Roche Diagnostics, Rotkreuz, Switzerland). These nitrocellulose membranes were blocked with 5% skimmed milk powder/Tris-buffered saline with 0.1% Tween20 (w/v) at 4 °C overnight. After that, the membranes were incubated with the primary antibodies:inhibitor of κB kinase-α (IκB-α; Santa Cruz Biotechnology, Delaware Ave Santa Cruz, CA, USA; dilution ratio; 1:1000), TLR4, CD14 (Santa Cruz Biotechnology, USA; dilution ratio; 1:200), p-p38, p-JNK, p-ERK, total p38, total JNK, total ERK (Cell Signaling Technology, Boston, CA, USA; all at 1:200), and β-actin (Cell Signaling Technology, Boston, CA, USA; 1:500); the samples were incubated overnight at 4 °C. Then, membranes were treated with HRP-conjugated anti-rabbit IgG (H + L) as the second antibody (Promega, Madison, WI, USA; dilution ratio; 1:2000). Chemiluminescent HRP Substrate examined the immunoblotting (Cat. NO: WBKLS0100; ImmobilonTMWestern, MA, USA) according to the manufacturer’s instructions. Membranes were exposed by a FUJIFILM Luminescent Image Analyzer LAS-1000 (Macintosh TM, USA). The intensities of the resulting bands were quantified by Quantity One software on an AGS-800 densitometer (BioRad, Hercules, CA, USA).
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4

Western Blot Analysis of STAT3 and STAT5

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Immobilon-P PVDF membranes (Millipore) were probed with the primary antibodies at 4°C overnight and then washed and incubated with HRP-conjugated goat anti-mouse IgG (H+L) or anti-rabbit IgG (H+L) secondary antibody (Promega). The following primary antibodies were used: anti-STAT3 (clone D3Z2G, Cell Signaling Technology), anti-phospho-STAT3 (Tyr705) (clone D3A7, Cell Signaling Technology), anti-STAT5 (Cell Signaling Technology), anti-phospho-STAT5 (Tyr694) (clone D47E7, Cell Signaling Technology), anti-β-actin (clone C4, Santa Cruz Biotechnology, sc-47778), HRP-conjugated anti-mouse IgG (H+L) (Promega, W4021), and HRP-conjugated anti-rabbit IgG (H+L) (Promega, W4011). All images were acquired with ChemiDoc MP system (Bio-Rad) and Image Lab software (Bio-Rad). Protein levels for each blot were quantified with ImageJ software.
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5

Western Blot Analysis of Liver Proteins

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Proteins from 50 mg of liver samples were extracted, separated in gradient SDS-PAGE gels and electroblotted onto nitrocellulose membranes and western blot analysis was performed as described49 (link). Specific blotted proteins were detected with the corresponding primary rabbit antibody: anti-AMPK, anti-phospho-AMPK, anti-Akt, -anti-phospho-Akt (Cell Signaling Technology Inc. MA.; cat. number: #2532, #2535, #9272 and #9271, respectively); mouse anti-βactin (Sigma-Aldrich; cat. number: # A5316) and anti-Adaptin γ (Abcam, Cambridge, UK.;, cat. number #ab167153) followed by 1 h incubation with the corresponding secondary antibody: HRP-conjugated anti-rabbit IgG (H+L) or HRP-conjugated anti-mouse IgG (H+L) antibodies (Promega; cat. number W4011 and W4021, respectively). Specific protein bands were revealed using the enhanced chemiluminiscence detection system (Santa Cruz, Biotechnology Inc. CA, USA), in accordance with the manufacturer’s instructions. Images were visualized in an Autochemi-UVP Bioimaging System. Finally, bands were quantified by densitometry performed by ImageJ software (http://imagej.nih.gov/ij). Levels of specific proteins were normalized to actin levels for liver samples or adaptin levels for skeletal muscle samples.
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6

DENV and ZIKV NS1 Protein ELISA

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Recombinant NS1 proteins from DENV-1-Nauru/Western Pacific/1974 (Accession: M23027.1), DENV-2-Thailand/16681/84 (U87411.1), DENV-3-Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (AY099336.1), DENV-4-Dominica/814669/1981 (AF326573.1), ZIKV-Suriname (AZS35340.1), and ZIKV-Uganda MR766 (AWF93629.1) produced in HEK293 cells were purchased from The Native Antigen Company (United Kingdom). Nunc Maxisorp plates (ThermoFisher Scientific) were coated with recombinant NS1 (50 μL/well of 2 μg/mL) in coating buffer (0.1M NaHCO3) overnight at 4°C. Wells were washed three times with PBST (PBS supplemented with 0.05% Tween) and blocked with PBS supplemented with 5% skim milk (Sigma-Aldrich) for 1 h at RT. After washing the wells once with PBST, purified antibodies (50 μL of 1μg/mL) was added into the wells for 2 h at RT. Wells were washed three times with PBST, and 50 μL/well of peroxidase-conjugated anti-human IgG (1/5000 dilution, Sigma Aldrich) or HRP-conjugated anti-rabbit IgG (H+L) (1/5000 dilution, Promega) was added for 2 h at RT. After the wells were washed 3X with PBST, 3,3’,5,5’-Tetramethylbenzidine (TMB) Liquid Substrate system for ELISA (50 μL/well, Sigma) was added for ~ 5minutes, and then 1M HCL was added. The OD of the wells were determined at 450nm and 650nm (reference OD).
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7

Quantifying hLF and N-lobe proteins

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PC-14 and PC-3 cells were treated with 0.38 μM N-lobe only or 0.38 μM N-lobe plus 0.38 μM CS-E at 37 °C for 1 h. After washing with PBS, the cells were treated with trypsin/EDTA for 3 min. Next, the hLF and N-lobe were analyzed via western blotting using an anti-hLF polyclonal antibody (1:10000, A80-144A, Bethyl Laboratories, Inc.) and HRP-conjugated anti-rabbit IgG (H+L) (1:10000, Promega Corporation, Madison, WI, USA). Endogenous controls were detected using an anti-γ-actin antibody (1:5000, FUJIFILM Wako Pure Chemical Corporation) and HRP-conjugated anti-mouse IgG (H+L) (1:10000, Promega Corporation). The relative band intensities were represented graphically using GraphPad Prism software version 8.4.1.
(San Diego, CA, USA).
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8

UV-induced ISG15 regulation assay

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The following chemical reagents were used at the indicated concentrations: ISRIB (Tocris), 200 nM; dithiothreitol (DTT; Sigma-Aldrich), 5 mM; and poly(I:C), 15 μg (Invivogen). Cells were irradiated using a Spectrolink UV crosslinker at energy setting 200 J/m2. The following primary antibodies were used: rabbit polyclonal anti-RPS10/eS10 (1:2000; Abclonal, A6056), rabbit polyclonal anti-ISG15 (1:5000; Cell Signaling Technology, 2743), rabbit polyclonal anti-ZNF598 (1:7000; Sigma-Aldrich, HPA041760), rabbit monoclonal anti-RPS20/uS10 (1:1000; Abcam, ab133776), rabbit monoclonal anti-phospho-eIF2α (Ser51) (1:2000; Cell Signaling Technology, D9G8) and mouse monoclonal anti-α-tubulin (1:10,000; Cell Signaling Technology, DM1A). The following secondary antibodies were used (at 1:5000 dilution): HRP-conjugated anti-rabbit IgG (H+L) (Promega, W4011) and HRP-conjugated anti-mouse IgG (H+L) (Promega, W4021). Cells were transfected using Lipofectamine RNAiMax (Thermo Fisher Scientific) for poly(I:C) transfections and with Lipofectamine 2000 (Thermo Fisher Scientific) for plasmid transfections. Cells were treated with indicated units of interferon-β for 16 h.
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