Small-scale immunoprecipitations were performed in parallel with the main IP to verify Numb IP. Proteins were separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked for 1 hr at 4°C in 50 mM Tris–HCl buffer, 0.15 M NaCl, 1% Tween-20 (TBS) supplemented with 0.5% non-fat dry milk (blocking solution) and incubated overnight at 4°C with a rabbit monoclonal anti-Numb antibody (Cell Signaling #2756; RRID:AB_2154298 ) diluted 1:1000 in TBS/5% BSA. After washing with TBS, the blot was incubated with HRP-conjugated anti-rabbit IgG (1:8000 dilution in blocking solution, Cytiva NA934; RRID:AB_772206 ), the blot developed with ECL Prime reagent (RPN2236, Cytiva) and imaged with an Amersham ImageQuant 800 imager (Cytiva).
Hrp conjugated anti rabbit igg
Manufactured by Cytiva
Sourced in Japan, United States
HRP-conjugated anti-rabbit IgG is a secondary antibody conjugated with Horseradish Peroxidase (HRP). It is used to detect and quantify rabbit primary antibodies in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime reagent, by Cytiva (3 mentions)
Amersham imagequant 800 imager, by Cytiva (3 mentions)
Amersham imagequant, by Cytiva (3 mentions)
Hrp conjugated anti mouse igg, by Cytiva (3 mentions)
Chloroquine, by Merck Group (2 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Amersham ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Anti lamin b1, by Proteintech (1 mentions)
β actin, by Merck Group (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Anti iκb, by Cell Signaling Technology (1 mentions)
Lysopure nuclear and cytoplasmic extractor kit, by Fujifilm (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Elisa plate, by Greiner (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Precision microplate reader, by Molecular Devices (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
11 protocols using hrp conjugated anti rabbit igg
1
Numb Protein Immunoprecipitation and Analysis
2
Numb Protein Immunoprecipitation
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Small scale immunoprecipitations were performed in parallel with the main IP to verify Numb immunoprecipitation. Proteins were separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked for 1 hr at 4 °C in 50 mM Tris HCl buffer, 0.15 M NaCl, 1% Tween-20 (TBS) supplemented with 0.5% non-fat dry milk (blocking solution), and incubated overnight at 4 °C with a rabbit monoclonal anti-Numb antibody (Cell Signaling #2756) diluted 1:1000 in TBS/5% BSA. After washing with TBS, the blot was incubated with HRP-conjugated anti-rabbit IgG (1:8,000 dilution in blocking solution, Cytiva), the blot developed with ECL Prime reagent (Cytiva) and imaged with an Amersham ImageQuant 800 imager (Cytiva).
3
Immunoblotting of Liver Fractions
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The livers were washed with cold PBS. Nuclear/cytosolic fractions were isolated using Lysopure™ Nuclear and Cytoplasmic Extractor Kit (Wako Chemical), according to the manufacturer's protocol. Ten micrograms of protein were loaded on 10% Mini-PROTEAN TGX gels (Bio-Rad) and transferred to PVDF membranes. The membranes were incubated with anti-IκB (1:1,000, catalog no. #4812, Cell Signaling Technology), anti-NF-κB p65 (1:1,000, catalog no. #8242, Cell Signaling Technology), anti-Lamin B1 (1:2,000, catalog No. 66095-1-lg, proteintech), β-actin (catalog no. A5441, Sigma-Aldrich), anti-Ido2 (1:1,000, catalog no. ab214214, abcam), anti-Ido1 (1:1,000, catalog no. MABF850, Merck Millipore, Darmstadt, Germany), or anti-Tdo2 (1:1,000, catalog no. MABN1537, Merck Millipore). The membranes were then incubated with HRP-conjugated anti-mouse IgG (1:10,000, catalog no. NA931A, Cytiva., Tokyo, Japan) or HRP-conjugated anti-rabbit IgG (1:5,000, catalog no. NA934V, Cytiva., Tokyo, Japan), respectively. The detection of target proteins was performed with Amersham ECL Prime Western Blotting Detection Reagent (Cytiva). Protein levels were quantified by ImageJ software (National Institutes of Health, Bethesda, MD). To re-probe the PVDF membranes, the antibodies bound to the membranes were removed by a commercial stripping solution.
4
Quantifying PfRipr Protein Levels by ELISA
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Ninety-six well enzyme-linked immunosorbent assay (ELISA) plates (Greiner Bio-One, Kremsmünster, Austria) were coated with 50 μL of mouse anti-PfRipr mAb (clone 29B11) (produced as described in supplementary information), diluted to 2.5 μg/ml in a catcher buffer (25 mM carbonate buffer pH 9.6) and incubated at 4°C overnight. The plates were washed with phosphate-buffered saline (PBS) with 0.1% (v/v) Tween-20 (PBS-T) and then blocked with 200 μL 3% skimmed milk in PBS-T for 1 h at RT. Purified PfRipr5 was diluted in PBS, added to antibody-coated plates thrice, and incubated for 1 h at RT. After washing, the plates were incubated with 100 μL of th anti-PfRipr5 rabbit antiserum diluted 1:2,000 in PBS for 1 h at RT. After incubation, the plates were washed and incubated with HRP-conjugated anti-rabbit IgG (Cytiva) diluted 1:1,000 in PBS-T for 1 h at RT. The plates were washed and incubated for 15 min at RT with the TMB substrate solution (Thermo Scientific). The reaction was stopped with 2 M sulfuric acid, and the optical density (OD) was determined at 450 nm using a precision microplate reader (Molecular Devices, Sunnyvale, CA).
5
Numb Protein Immunoprecipitation Protocol
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Small scale immunoprecipitations were performed in parallel with the main IP to verify Numb IP. Proteins were separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked for 1 hr at 4 °C in 50 mM Tris HCl buffer, 0.15 M NaCl, 1% Tween-20 (TBS) supplemented with 0.5% non-fat dry milk (blocking solution), and incubated overnight at 4 °C with a rabbit monoclonal anti-Numb antibody (Cell Signaling #2756) diluted 1:1000 in TBS/5% BSA. After washing with TBS, the blot was incubated with HRP-conjugated anti-rabbit IgG (1:8,000 dilution in blocking solution, Cytiva), the blot developed with ECL Prime reagent (Cytiva) and imaged with an Amersham ImageQuant 800 imager (Cytiva).
6
Characterization of Brain-Derived Amyloid-β Oligomers
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Human brain‐derived Aβo was characterized by Western blot analysis. Two different concentrations of Aβo (1 and 0.5 µg of protein) were loaded onto precast NuPAGE 4–12% Bis‐Tris gel (Invitrogen) for SDS‐PAGE analysis. The gel was subsequently transferred onto nitrocellulose membranes and blocked with 10% nonfat dry milk at 4℃ overnight. The membrane was then probed with primary antibodies, A11 (1:1000) and anti‐Aβ1−17 6E10 (1:6000, BioLegend, USA) diluted in 5% nonfat dry milk for 1 h at RT. HRP‐conjugated anti‐rabbit IgG and anti‐mouse IgG (1:6000, Cytiva) were used to detect A11 and 6E10 immunoreactivity, respectively. ECL plus (Cytiva) was used to visualize the bands.
7
Autophagy Regulation in Cell Cultures
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Minimum essential medium (α-MEM) and Dulbecco modified minimal essential medium (D-MEM) was obtained from Gibco Laboratories (Buenos Aires, Argentina). Fetal bovine serum (FBS) was purchased from Natocor S.A. (Córdoda, Argentina). The inhibitors α-difluoromethylornithine (DFMO) and Chloroquine (CQ) and the TRITC-conjugated phalloidin was purchased from Sigma (Buenos Aires, Argentina). The rabbit anti-Beclin-1 antibody was purchased from Santa Cruz (Santa Cruz Biotechnology, INC) and the polyclonal rabbit anti-LC3 antibody from Sigma (Buenos Aires, Argentina). The following antibodies were also used: anti-Atg5 (Abcam), anti-p62 (Abcam), Anti-ubiquitin (Santa Cruz), anti-β-actin (Genescript) and the anti-β tubulin (E7) was obtained from Developmental Studies Hybridoma Bank. The secondary antibodies Cy3-conjugated anti-mouse and Cy3-conjugated anti-rabbit were purchased from Jackson Immuno Research Laboratories, INC, as well as the antibodies HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG and HRP-conjugated Anti-goat IgG. Hybond-ECL nitrocellulose membranes were from Amersham. The DNA marker Hoechst 33342 was purchased from Life Technologies.
8
Antibody Characterization for Protein Detection
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Mouse monoclonal anti-TFRC antibody (Zymed, 136800), rabbit polyclonal cleaved CASP3 (Asp-175) antibody (Cell Signaling Technology, 9664), rabbit polyclonal anti-ATG5 antibody (Cell Signaling Technology, 8540), rabbit monoclonal anti-phospho-TBK/NAK (Ser172) antibody (Cell Signaling Technology, 5483), rabbit monoclonal anti-TBK1/NAK antibody (Cell Signaling Technology, 3504 and Abcam, ab40676), mouse monoclonal anti-HA antibody (Roche Applied Biosystems, 11583816001), rabbit polyclonal anti-HA (Santa Cruz, sc-805), rabbit polyclonal and mouse monoclonal anti-GFP (Santa Cruz, sc-9996, sc-8334), anti-GAPDH (Millipore, MAB374) and anti-actin (Millipore, MAB1501) are commercially available. Secondary antibodies used were Cy-3-conjugated anti-mouse IgG (Amersham, PA43002), Cy-3-conjugated anti-rabbit IgG (Amersham, PA43004), HRP conjugated anti-mouse IgG (Amersham, NA9310), HRP conjugated anti-rabbit IgG (Amersham, NA934), Alexa-488 anti-mouse and anit-rabbit IgG (Molecular Probes, A21202, A21206). Phospho-OPTN antibody specific to S177 residue of OPTN was a generous gift from Dr. Ivan Dikic of Goethe University Medical School, Theodor-Stern-Kai 7 60590 Frankfurt am Main / Germany [27 (link)]. BX-795 (Calbiochem, 204001) and chloroquine (Sigma, C6628) are commercially available.
9
Immunohistochemical Detection of Neutrophil Extracellular Traps
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Deparaffinisation and rehydration were performed with standard xylene-to-ethanol washes. Heat-induced epitope retrieval was achieved by boiling samples for 30 min in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Samples were blocked in phosphate buffered saline plus 0.025% Tween 20 with 10% foetal bovine serum. The primary antibody was to MPO (Dako, Carpinteria, California, USA), while the secondary antibody was HRP-conjugated anti-rabbit IgG (Amersham Biosciences, Pittsburgh, Pennsylvania, USA). In between the primary and secondary antibodies, tissue was incubated for 15 min in 0.3% hydrogen peroxide to block endogenous peroxidase activity. Colour change was detected with the DAB-Plus Substrate Kit (Invitrogen, Grand Island, New York, USA). Samples were counterstained with haematoxylin. For a subset of the samples, the identity of netting neutrophils was confirmed (as MPO-DNA overlap) by immunofluorescence staining with anti-MPO (Dako) and DAPI as previously described.30
10
Western Blot Analysis of Nrf2 Pathway
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Cells were lysed in 1× Laemmli buffer and centrifuged at 10,000 g for 2 min through a Nucleo Spin filter (Machery Nagel). Lysates were boiled at 95 °C for 5 min, 20 μg of total protein were subjected to separation by a 10% SDS gel, transferred onto nitrocellulose membranes (Amersham Biosciences) and blocked with 5% milk powder in PBS Tween (0.1%) for 1 h. The following antibodies were used: anti-Nrf2 (monoclonal, 1:1000, Santa Cruz); anti-GCLC (rabbit, 1:1000, Bioworld Technologies); anti-GCLM (rabbit, 1:2000, Proteintech); anti-CYPOR (monoclonal, 1:500, Santa Cruz); HRP-conjugated anti mouse IgG (1:5000, Jackson Immuno Research); and HRP-conjugated anti-rabbit IgG (1:5000, Amersham). Protein bands were detected by a FUSION SL™ system (Peqlab, Erlangen, Germany) and quantified by ImaEva or ImageJ.
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