The total protein was extracted from log-phase cells with an NuPAGE LDS sample buffer (ThermoFisher) after 0.2N NaOH treatment (Kushnirov, 2000 (link)). For each analysis, the total protein extracted from two optical density (OD) units of cells with OD600 was used. For total protein visualization, the extracted total protein was labeled with Ezlabel FluoroNeo (ATTO), as described in the manufacturer’s protocol, and separated by 4–12% SDS-PAGE. Proteins were detected and measured using the LAS-4000 image analyzer (GE Healthcare) in SYBR–green fluorescence detection mode and Image Quant TL software (GE Healthcare). The expression of each target protein (AU) was calculated, as shown in Figure 2 . Average values, SD, and p-values of Welch's t-test were calculated from biological triplicates. For detection of Tpi1, the SDS-PAGE-separated proteins were transferred to a PVDF membrane (ThermoFisher). Tpi1 was detected using an anti-Tpi1 antibody (RRID:AB_11130951 ), a peroxidase-conjugated secondary antibody (Nichirei Biosciences), and a chemiluminescent reagent (ThermoFisher). The chemiluminescent image was acquired with an LAS-4000 image analyzer in chemiluminescence detection mode.
Peroxidase conjugated secondary antibody
Manufactured by Nichirei Biosciences
Sourced in Japan
Peroxidase-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It is composed of a secondary antibody labeled with the enzyme peroxidase, which can catalyze a color-producing reaction when exposed to a suitable substrate. This product is primarily used to detect and amplify the signal from primary antibodies in applications such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Nupage lds sample buffer, by Thermo Fisher Scientific (4 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Las 4000 image analyzer, by Fujifilm (3 mentions)
Iblot transfer stack pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Anti gfp antibody, by Roche (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Las 4000 image analyzer, by GE Healthcare (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
3 30 diaminobenzidine, by Agilent Technologies (1 mentions)
Immpact dab substrate kit, by Vector Laboratories (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
Bz x710, by Keyence (1 mentions)
Leupeptin, by Peptide Institute (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
8 protocols using peroxidase conjugated secondary antibody
1
Quantitative Protein Analysis Protocol
2
Western Blot Detection of TAP-tagged Proteins
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Detection of TAP-tag protein by Western blot was performed as described in Ishikawa et al.37 (link). Yeast strains were aerobically cultured at 30 °C in 2 mL of YPD medium. Optical density at 660 nm (OD660) was measured, and units of 1 OD660 were harvested during the log phase. Cells were treated with 1 mL of 0.2 N NaOH for 5 min at room temperature and then suspended in 2× NuPAGE LDS sample buffer (Invitrogen) and heated to 70 °C for 10 min. Protein lysate in the supernatant was labeled with EzLabel FluoroNeo (ATTO) and subjected to polyacrylamide gel electrophoresis with lithium dodecyl sulfate (SDS-PAGE), followed by Western blot with PAP (Sigma-Aldrich) (1:2000) and peroxidase-conjugated secondary antibody (Nichirei Biosciences) (1:1000). We used a NuPAGE 4–12% Bis-Tris Gel (Invitrogen) for SDS-PAGE and an iBlot Transfer Stack PVDF membrane (Invitrogen) for Western blot. Chemiluminescence was induced by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and detected on a LAS-4000 image analyzer (Fujifilm) using ImageQuant LAS 4000 (GE Healthcare).
3
Quantifying Yeast Protein Levels
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Yeast cells were grown in 2 mL of the appropriate medium and subcultured in 3 mL of fresh medium. The optical density at 600 nm (OD600) was measured and 2 OD600 units were harvested at log-phase. The cells were treated with 1 mL of 0.2 N NaOH for 5 min at room temperature and then were suspended in 2× NuPAGE LDS Sample Buffer (Invitrogen) and heated at 70°C for 10 min. The supernatant corresponding to 0.5 OD600 units was labeled with EzLabel FluoroNeo (ATTO) and subjected to polyacrylamide gel electrophoresis with lithium dodecyl sulfate (SDS-PAGE), followed by Western blotting with PAP (Sigma-Aldrich) (1:2000) or an anti-GFP antibody (Roche) (1:1000) and peroxidase-conjugated secondary antibody (Nichirei Biosciences) (1:1000). We used NuPAGE 4%–12% Bis-Tris Gel (Invitrogen) for SDS-PAGE and iBlot Transfer Stack PVDF membrane (Invitrogen) for Western blotting. Chemiluminescence was induced by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and detected using LAS-4000 image analyzer (Fujifilm) and ImageQuant LAS 4000 (GE Healthcare). The band intensity was quantified using ImageQuant TL (GE Healthcare), and the fold change of protein levels was calculated as shown in S2 Fig according to a previously described method [11 (link)].
4
Western Blot Detection of GFP Expression
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The detection of GFP was performed by Western blot as previously described [78 (link)]. ENA1-GFP cells were grown in YPD, 1 M NaCl/YPD, and 1 M NaCl/YPD with 5 mM CaCl2. The cells were harvested at the log phase (OD660 = 1.0) and treated with 1 ml of 0.2 M NaOH. 50 μl of 1xNuPAGE LDS sample buffer (Invitrogen, USA) was added, and the mixture was heated at 70°C for 10 minutes. The protein lysate was labeled with Ezlabel FluoroNeo (ATTO, Japan) and separated by polyacrylamide gel electrophoresis on 4–12% NuPAGE 4%–12% Bis-Tris Gel (Invitrogen, USA). The separated proteins were transferred onto a PVDF membrane (Invitrogen, USA) using the iBlot (Invitrogen, USA). GFP was probed using an anti-GFP antibody (Roche) (1:1,000) and a peroxidase-conjugated secondary antibody (Nichirei Biosciences, Japan) (1:1,000) followed by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA). The band intensity was detected and measured using the LAS-4000 image analyzer (Fujifilm, Japan) and quantified using ImageJ (1.53k).
5
Immunohistochemical Analysis of Kidney Tissue
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Kidneys were incubated in 10% formalin, embedded in paraffin, and then sectioned at a thickness of 1 mm (periodic acid-Schiff reagent staining) or 4 mm (immunohistochemistry). The sections were stained with mouse monoclonal anti-human CD147 antibody (Ab; Abcam, Cambridge, MA), rabbit monoclonal anti-human b 1 integrin Ab (Abcam), goat antimouse Bsg Ab (R&D Systems), and rabbit anti-mouse WT-1 Ab (Santa Cruz Biotechnology, Dallas, TX), followed by detection using a peroxidase-conjugated secondary antibody (Nichirei, Tokyo, Japan). The staining was visualized with 3,3 0 -diaminobenzidine (Dako, Carpinteria, CA), which produces a brown color. Podocytes positive for WT-1 were counted by examining all glomeruli of the cortex under a microscope at high magnification. Quantitation was performed by two experts (T.M. and K.M.) in nephropathology who were blinded to the experimental conditions. For transmission electron microscopic analysis, kidneys were fixed in formalin, embedded in epoxy resin, sectioned, and then stained with uranyl acetate and lead citrate. As described by Koop et al, 32 the total length of the GBM and the number of slit pores were evaluated in the free capillary wall, and then the average foot process width was examined by calculating the ratio of the total length of the GBM to the total number of slits.
6
Immunohistochemistry of TGFBR2 and FOXO3
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IHC was conducted to evaluate the protein levels of TGFBR2 and FOXO3. Surgically collected pieces of kidney tissue were fixed in 4% paraformaldehyde and then embedded in paraffin. The tissues were sectioned at 4 µm thick, deparaffinized, and rehydrated. After antigen activation and blocking, kidney sections were incubated overnight at 4 °C with a TGFBR2-specific primary antibody (Fitzgerald Industries International, Acton, MA, USA) and FOXO3-specific primary antibody (Bethyl, Waltham, MA, USA). After washing, they were incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature (NICHIREI, Tokyo, Japan). Then, kidney sections were incubated with ImmPACT DAB Substrate Kit (Vector, Newark, CA, USA) for 10 min at room temperature, incubated in Meyer hematoxylin solution (Muto Pure Chemicals) for 10 s, washed, and dehydrated. Kidney sections were observed using a BZ-X710 (Keyence). The IHC positive area (brown stain) was quantified. Six images from random fields were obtained at 200× magnification. The brown stained area was measured using Keyence Hybrid Cell Count on a BZ-X Analyzer (version 1.4.1.1, Keyence). For randomization, digital images of kidney sections were segmented, numbered in sequence, and selected using a random number generator [https://www.random.org/ (accessed on 15 October 2022)].
7
Endogenous CAPN Activation Assay
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Harvested cells were lysed in TED buffer [10 mM Tris/Cl (pH 7.0), 1 mM EDTA, 1 mM DTT] including protease inhibitors [2 mM PMSF (Sigma-Aldrich), 0.1 μM leupeptin (Peptide Institute), 40 μM bestatin (Sigma-Aldrich), 1.5 μM aprotinin (Sigma-Aldrich), and 0.7 μM CAST (Takara Bio)] using 27G syringe needles. For immunoblot, samples were boiled in SDS-sample buffer. For endogenous CAPN activation assay, cell lysates prepared as above were incubated with 10 mM EDTA, 10 mM CaCl2, 150 mM NaCl, or 0.7 µM CAST for 30 min at 30°C (Ono et al., 2010 (link)). SDS-PAGE and immunoblot analyses were performed as previously described (Ojima et al., 2007 (link)). Antibodies used in this study were listed in Table 1 . Band signals were visualized using peroxidase-conjugated secondary antibodies (Nichirei Bioscience) and a POD immunostaining set (FUJI FILM Wako Pure Chemical).![]()
8
Characterization of Epidermal Equivalents
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The fixed epidermal equivalents of cultured keratinocytes derived from hESCs and postnatal epidermis by air-lifting culture were routinely processed into 5 μm thick paraffin-wax-embedded sections and haematoxylin and eosin (HE) staining was performed using a standard protocol. Expressions of E-cadherin (CDH1), pan-keratins (KRTs), KRT14, keratin10 (KRT10), IVL, TP63, and vimentin (VIM) were analyzed by immunohistological methods. The sections were treated with mouse monoclonal anti-CDH1 (1:100, 36/E-Cadherin, Becton Dickinson, NJ, USA), mouse monoclonal anti-KRTs (1:100 dilution, AE1/AE3, Thermo Fisher Scientific), mouse monoclonal anti-KRT14 (1:100, LL002, Abcam, Cambridge, UK), rabbit polyclonal anti-KRT10 (1:100, BioLegend, CA, USA), mouse monoclonal anti-IVL (1:1000, SY5, Sigma–Aldrich), mouse monoclonal anti-TP63 (1:100, 4A4, Abcam), and mouse monoclonal anti-VIM (1:100, Vim 3B4, Dako, Agilent Technologies, CA, USA) at room temperature for 90 min and were stained with peroxidase-conjugated secondary antibodies (Nichirei Bioscience, Tokyo, Japan) in accordance with the manufacture's suggested protocol. Human dermal and epidermal tissues were used for negative control of immnohistological analysis, and the results are shown in Supplemental Fig. 2 .
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