Hrp immunstar detection kit

Manufactured by Bio-Rad

The HRP Immunstar detection kit is a substrate-based chemiluminescent detection system designed for the visualization of horseradish peroxidase (HRP)-labeled proteins in Western blotting applications. The kit provides a sensitive and reliable method for the detection of target proteins.

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Lab products found in correlation

Image station 440cf, by Kodak (4 mentions) Pvdf membrane, by Bio-Rad (3 mentions) 1d image software, by Kodak (3 mentions) Glut5, by Merck Group (1 mentions) Glut5, by Cell Signaling Technology (1 mentions) Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)

4 protocols using hrp immunstar detection kit

Western Blot Analysis of Metabolic Enzymes

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Protein lysates were prepared from mouse tissue employing MAP Kinase lysis buffer as previously described(Lanaspa et al., 2007 (link)). Protein content was determined by the BCA protein assay (Pierce). Total protein (50 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% w/v), and transferred to PVDF membranes (BioRad). Membranes were first blocked for 1 h at 25 °C in 4% (w/v) instant milk dissolved in 0.1 % Tween-20 Tris-Buffered Saline (TTBS), incubated with primary rabbit or mouse-raised antibodies (1:1000 dilution in TTBS) KHK (Sigma HPA007040; RRID:AB_1079185), KHK-A (SAB Signalway; Cat# 21708), KHK-C (SAB Signalway; Cat# 21709), Glut5 (Millipore, 07–1406 lot 2999837, this antibody and lot has been validated using specific Glut5 KO mice, a representative western blot is shown in supplemental figure 2) and Actin (Cell Signaling 4968; RRID:2313904) and visualized using an anti-rabbit (7074; RRID:AB_2099233) or anti-mouse IgG (7076; RRID:AB_330924) horseradish-peroxidase conjugated secondary antibody (1:2000, Cell Signaling) using the HRP Immunstar® detection kit (Bio-Rad, Hercules, CA). Chemiluminescence was recorded with an Image Station 440CF and results analyzed with the 1D Image Software (Kodak Digital Science, Rochester, NY).

Andres-Hernando A., Orlicky D.J., Kuwabara M., Ishimoto T., Nakagawa T., Johnson R.J, & Lanaspa M.A. (2020). Deletion of fructokinase in the liver or in the intestine reveals differential effects on sugar-induced metabolic dysfunction. Cell metabolism, 32(1), 117-127.e3.

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Western Blot Analysis of Muscle Signaling Proteins

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Protein lysates were prepared from mouse tissue or C2C12 cells using lysis buffer containing 0.3% Triton X-. Protein content was determined by the BCA protein assay (Pierce, Rockford, IL). Total protein (50 μg) was separated by SDS-PAGE [10% (w/v)] and transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were first blocked for 1 hat 25°C in 4% (w/v) instant milk dissolved in 0.1% Tris-buffered saline with Tween 20 TBS (TTBS) and incubated with the following primary rabbit-raised antibodies (1:1,000 dilution in TTBS): Myostatin (19142-1-AP, Proteintech; RRID:AB_10638615), AMPD1 (19780-1-AP; RRID:AB_10644281), GLUL (80,636, Cell Signaling; RRID:AB_2799956), pIRS1^Ser636/639 (2388, Cell Signaling; RRID:AB_330339), IRS1 (2382, Cell Signaling; RRID:AB_330333), pAKT^Ser473 (4058 Cell Signaling; RRID:AB_331168), AKT (9272, Cell signaling; RRID:AB_32982) and GAPDH (5174, Cell Signaling; RRID:AB_10622025) and visualized using an anti-rabbit (no. 7074) horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, Cell Signaling) using the HRP Immunstar detection kit (Bio-Rad). Chemiluminescence was recorded with an Image Station 440CF, and results were analyzed with the 1D Image software (Kodak Digital Science, Rochester, NY). Data for proteins of interest are expressed normalized to GAPDH expression.

Andres-Hernando A., Cicerchi C., Garcia G.E., Orlicky D.J., Stenvinkel P., Johnson R.J, & Lanaspa M.A. (2023). Phosphate depletion in insulin-insensitive skeletal muscle drives AMPD activation and sarcopenia in chronic kidney disease. iScience, 26(4), 106355.

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Protein Extraction and Western Blotting from Mouse Tissues

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Protein lysates were prepared from mouse tissue employing MAP Kinase lysis buffer as previously described (47 (link)). Protein content was determined by the BCA protein assay (Pierce). Total protein (50 μg) was separated by SDS-PAGE (10% w/v) and transferred to PVDF membranes (BioRad). Membranes were first blocked for 1 hour at 25°C in 4% (w/v) instant milk dissolved in 0.1% Tween-20 Tris-Buffered Saline (TTBS); incubated with primary rabbit or mouse-raised antibodies (1:1000 dilution in TTBS) KHK (Sigma, HPA007040; RRID: AB_1079185), FAS (Cell Signaling, 3180; RRID: AB_2100796), ACC (Cell Signaling, 3676; RRID: AB_2219397), V1bR (BIOSS; bs-11800R), and actin (Cell Signaling, 4968; RRID: 2313904); and visualized using an anti-rabbit (7074; RRID: AB_2099233) or anti-mouse IgG (7076; RRID: AB_330924) horseradish peroxidase–conjugated secondary antibody (1:2000, Cell Signaling) using the HRP Immun-Star Detection Kit (Bio-Rad). Chemiluminescence was recorded with an Image Station 440CF and results analyzed with the 1D Image Software (Kodak Digital Science). See complete unedited blots in the supplemental material.

Andres-Hernando A., Jensen T.J., Kuwabara M., Orlicky D.J., Cicerchi C., Li N., Roncal-Jimenez C.A., Garcia G.E., Ishimoto T., Maclean P.S., Bjornstad P., Sanchez-Lozada L.G., Kanbay M., Nakagawa T., Johnson R.J, & Lanaspa M.A. (2021). Vasopressin mediates fructose-induced metabolic syndrome by activating the V1b receptor. JCI Insight, 6(1), e140848.

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Western Blot Analysis of Mouse Tissue Proteins

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Protein lysates were prepared from mouse tissues using MAP kinase lysis buffer as described previously^{38 (link)}. Protein content was determined using the bicinchoninic acid protein assay (Pierce). Total protein (40 μg in liver and 25 μg in hypothalamic samples) was separated by SDS–polyacrylamide gel electrophoresis (10% w/v) and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories). Membranes were first blocked for 1 h at 25 °C in 4% (w/v) instant milk dissolved in 0.1% Tween 20 Tris-buffered saline, incubated with primary rabbit or mouse-raised antibodies (1:1,000 dilution in Tween 20 Tris-buffered saline) pSTAT3/STAT3 (catalogue no. 12640S, Cell Signaling Technology; research resource identifier (RRID): AB_2629499, AB_331586), AMPD2 (Abnova 271, RRID: AB_1236647) and actin (catalogue no. 4968S, Cell Signaling Technology; RRID: 2313904) and visualized using an anti-rabbit (catalogue no. 7074; RRID: AB_2099233) or anti-mouse IgG (catalogue no. 7076; RRID:AB_330924) horseradish peroxidase-conjugated secondary antibody (1:2,000, Cell Signaling Technology) using the HRP Immunstar detection kit (Bio-Rad Laboratories). Chemiluminescence was recorded with an Image Station 440 CF and results were analysed with the 1D Image software version 3.6 (Kodak Digital Science).

Andres-Hernando A., Cicerchi C., Kuwabara M., Orlicky D.J., Sanchez-Lozada L.G., Nakagawa T., Johnson R.J, & Lanaspa M.A. (2021). Umami-induced obesity and metabolic syndrome is mediated by nucleotide degradation and uric acid generation. Nature metabolism, 3(9), 1189-1201.

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