Expression of HSL, its serine-563-phosphorylated form (HSL Ser563) and ATGL were measured in epididymal fat pad homogenates by western blot. Antibodies for immunoblotting were as follows: anti-HSL (1:2000, Cell Signaling Technology, USA), anti-HSL Ser563 (1:500, Cell Signaling Technology), anti-ATGL (1:2000, Cell Signaling Technology) and anti-β-actin (1:5000, Sigma). Epididymal adipose tissue homogenates (25 µg total protein per well) were separated by SDS page, and proteins transferred to a polyvinylidene fluoride membrane. Membranes were then blocked [5% milk and 0.1% TWEEN-20 in PBS (PBS-T)], and incubated at 4°C overnight in primary antibodies diluted in 1% BSA in PBS-T. After being incubated with HRP-conjugated secondary antibodies (1:500, Wako Chemicals) in 5% milk in PBS-T, blots were developed using an Enhanced Chemiluminescent Detection Kit (GE Healthcare). Digital images of protein bands were acquired (ImageQuant 350, GE Healthcare, UK) and semi-quantitative analysis of protein content performed by densitometry using ImageQuant TL software (GE Healthcare, UK), with values normalised to β-actin as a loading control.
Hrp conjugated secondary antibodies
Manufactured by Fujifilm
HRP-conjugated secondary antibodies are laboratory reagents used in various immunoassay techniques, such as ELISA and Western blotting. They are designed to detect and amplify the signal from primary antibodies bound to target proteins or antigens. The horseradish peroxidase (HRP) enzyme conjugated to the secondary antibody catalyzes a colorimetric or chemiluminescent reaction, allowing for the visualization and quantification of the target of interest.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescent detection kit, by GE Healthcare (3 mentions)
Imagequant tl software, by GE Healthcare (3 mentions)
Imagequant 350, by GE Healthcare (3 mentions)
Anti β actin, by Merck Group (3 mentions)
β actin, by GE Healthcare (2 mentions)
Anti atgl, by Cell Signaling Technology (1 mentions)
Anti hsl, by Cell Signaling Technology (1 mentions)
Anti glut1, by Merck Group (1 mentions)
Anti claudin 5, by Abcam (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
4 protocols using hrp conjugated secondary antibodies
1
Western Blot Analysis of Adipose Tissue Proteins
2
Quantifying GLUT1 Protein Levels in Ischemic Brain
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Antibodies for immunoblotting were as follows: anti-GLUT1 (1:1000, Merck Millipore), anti-β-actin (1:5000, Sigma). Homogenised ipsilateral (ischaemic, I) and contralateral (non-ischaemic, NI) brain samples (25 µg of protein for each sample) were separated by SDS page, and proteins transferred to a polyvinylidene fluoride membrane. After being blocked (5% milk and 0.1% TWEEN-20 in PB-Saline (PBS-T)), membrane was incubated at 4℃ overnight in primary antibodies diluted in 1% BSA in PBS-T. Membranes were then incubated with HRP-conjugated secondary antibodies (1:500, Wako Chemicals) in 5% milk in PBS-T and blots developed using an Enhanced Chemiluminescent Detection Kit (GE Healthcare). Digital images of protein bands were acquired (ImageQuant 350, GE Healthcare, UK) and semi-quantitative analysis of protein content performed by densitometry using ImageQuant TL software (GE Healthcare, UK), with β-actin used as a loading control.
3
Immunoblot and Immunoprecipitation Protocols
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Regarding the immunoblot analysis, cultured cells or tissues were homogenized in RIPA buffer containing phosphatase and protease inhibitor cocktails. Equal amounts of protein were separated by SDS-PAGE, followed by immunoblotting. Immunoreactivity was visualized using HRP-conjugated secondary antibodies and a chemiluminescent substrate (Wako Pure Chemical Industries). Chemiluminescent images were captured by LAS4000-mini and quantified using ImageJ software. Scans at multiple exposures were obtained to ensure that the results fell within the linear range of the instrument.
Regarding immunoprecipitation, cells or tissues were lysed in TNE buffer (1% Triton X-100, 100 mM NaCl, and 20 mM Tris-HCl, pH 7.5) containing protease inhibitor cocktail. After centrifugation at 15,000 g for 30 min, the supernatant was incubated at 4°C overnight with the primary antibody. After being incubated with protein A-coupled Dynabeads, proteins were eluted by boiling for 5 min in Laemmli sample buffer and analyzed by immunoblotting.
Regarding immunoprecipitation, cells or tissues were lysed in TNE buffer (1% Triton X-100, 100 mM NaCl, and 20 mM Tris-HCl, pH 7.5) containing protease inhibitor cocktail. After centrifugation at 15,000 g for 30 min, the supernatant was incubated at 4°C overnight with the primary antibody. After being incubated with protein A-coupled Dynabeads, proteins were eluted by boiling for 5 min in Laemmli sample buffer and analyzed by immunoblotting.
4
Western Blot Analysis of Tight Junctions
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Antibodies for immunoblotting were as follows: anti-claudin-5 (1:1000, Abcam), anti-occludin (1:500, Life Technologies) and anti-β-actin (1:5000, Sigma). Microvessel samples were homogenised in buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 0.02% NaN3, 1% Triton X-100), separated by SDS page and proteins transferred to a polyvinylidene fluoride membrane. After being blocked (5% milk and 0.1% TWEEN-20 in PB-Saline (PBS-T)), membranes were incubated at 4℃ overnight in primary antibodies diluted in 1% BSA in PBS-T. Membranes were then incubated with HRP-conjugated secondary antibodies (1:500, Wako Chemicals) in 5% milk in PBS-T and blots developed using an Enhanced Chemiluminescent Detection Kit (GE Healthcare). Digital images of protein bands were acquired (ImageQuant 350, GE Healthcare, UK) and semi-quantitative analysis of protein content performed by densitometry using ImageQuant TL software (GE Healthcare, UK), with β-actin used as a loading control.
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