Frozen specimens from rodents were homogenized in 100 μl lysis buffer containing a mixture of proteinase and phosphatase inhibitors and then centrifuged at 15 000 rpm for 15 min at 4°C. The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL). A total of 50 μg of protein was resolved on 12% precasted SDS‐PAGE gels, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF membrane was blocked with 5% non‐fat milk in PBS containing 0.1% Tween 20 for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies. The following antibodies were used in this study: GAPDH, anti TNF‐α, anti‐ASIC1, and anti‐ASIC3 antibody (1:1000, abcam, USA). After washing with Tris‐Buffered Saline, 0.1% Tween 20 Detergent (TBST), the blots were incubated for 2 h at room temperature with HRP‐conjugated secondary antibody (1:5000; Amersham Biosciences, San Francisco, CA, USA), visualized by using Electro‐Chemi‐Luminescence (ECL) chemiluminescent detection system (Amersham Biosciences).
Hrp conjugated secondary antibody
Manufactured by Cytiva
Sourced in United States, United Kingdom
HRP-conjugated secondary antibodies are laboratory reagents used for detection and visualization in various immunoassays. They consist of a secondary antibody that is chemically conjugated to the enzyme horseradish peroxidase (HRP). The HRP enzyme catalyzes a colorimetric reaction, enabling the detection and quantification of target proteins or molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (9 mentions)
Pvdf membrane, by Merck Group (4 mentions)
β actin, by Merck Group (4 mentions)
Las 4000 imaging system, by Fujifilm (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Protein assay kit, by Bio-Rad (4 mentions)
Electro chemi luminescence chemiluminescent detection system, by Cytiva (3 mentions)
Gapdh, by Abcam (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Immobilon pvdf membrane, by Merck Group (3 mentions)
Pvdf membrane, by Cytiva (3 mentions)
Hybond ecl nitrocellulose membrane, by Cytiva (3 mentions)
Ecl plu, by Cytiva (3 mentions)
Hyperfilm, by Cytiva (3 mentions)
Protease and phosphatase inhibitor, by Merck Group (3 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (3 mentions)
Anti asic1, by Abcam (2 mentions)
Anti asic3 antibody, by Abcam (2 mentions)
Anti tnf α, by Abcam (2 mentions)
87 protocols using hrp conjugated secondary antibody
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of LC3B Protein Expression
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Cells cultured at 1 g and in RPM condition for 24 and 48 hours were lysed in RIPA buffer (Sigma-Aldrich). Samples were then clarified by centrifugation at 10000 rpm for 10 min. Equivalent amount of protein (10 μg) from each sample was electrophoretically resolved on 12.5% precast SDS-polyacrylamide gels (ExcelGel, GE Healthcare Biosciences) using horizontal apparatus (Pharmacia Biotech, Uppsala, Sweden). Then, separated proteins were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell) by a semidry system (Novablot, Pharmacia Biotech). Membranes were blocked with 3% nonfat milk in PBS and then were incubated (ON at 4°C) with the LC3B monoclonal antibody (1 : 2000; Sigma). After extensive washing with PBS containing 0.1% tween-20 (TBST), blots were incubated with 1 : 2000 dilution of HRP-conjugated secondary antibody (Amersham Biosciences) for 1 hour at RT. Immunopositive bands were detected with a chemiluminescence's detection system (GE Healthcare Biosciences). To check for equal loading of the gel, membranes were stripped and reprobed with mouse anti-β-actin antibody (1 : 20000, Sigma) and with anti-GAPDH antibody (1 : 1000, Cell Signalling Technology). Densitometric analysis was performed with the Quantity One software (BioRad Laboratories).
3
Protein Quantification and Immunoblotting
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Treated and untreated cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2, Sigma). Samples were clarified by centrifugation at 1000 rpm for 5 min. Equivalent amount of protein (10 μg) from each sample was electrophoretically resolved on 12.5% precast SDS-polyacrylamide gels (ExcelGel, GE Healthcare Biosciences) using horizontal apparatus (Pharmacia Biotech, Uppsala, Sweden). Then, separated proteins were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell) by a semidry system (Novablot, Pharmacia Biotech). Membranes were blocked with 5% BSA in PBS and then were incubated (overnight at 4°C) with the following monoclonal antibodies: GSK3α/β (Sigma), phospho-GSK3α/β (Ser 21/9) (cell signalling). After extensive washing with PBS containing 0.1% tween-20 (TBST), blots were incubated with 1 : 2000 dilution of HRP-conjugated secondary antibody (Amersham Biosciences) for 1 hour at room temperature. Immunopositive bands were detected with a chemiluminescence's detection system (GE Healthcare Biosciences). To check for equal loading of the gel, membranes were stripped and reprobed with mouse anti-β-actin antibody (1 : 20000, Sigma). Densitometric analysis was performed with the Quantity One software (BioRad Laboratories).
4
Western Blotting of HIF-1α in cDCs
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Western blotting was performed as describe in15 (link). Protein lysates were prepared from purified cDCs using RIPA buffer with phosphatase and protein inhibitors (Pierce). Proteins were separated with SDS-PAGE and transferred to Immobilon™ PVDF membrane (Millipore). Membranes were blocked for 1 h at room temperature in PBST containing 5% (w/v) milk, incubated overnight at 4 °C with anti-Hif1α antibody (Cell signalling) and subsequently with HRP-conjugated secondary antibody (Amersham Bioscience). Blotted proteins were detected using an EZ-ECL™ HRP chemiluminescence detection kit (Biological Industries) and exposed on to Hyperfilm™ photo film (Amersham).
5
Protein Extraction and Western Blot Analysis
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Total protein was extracted from lysed samples (20 µg) of the diabetic mouse lens capsule or the LECs of the cultured capsular bag under different treatment conditions and run on 10–15% SDS-PAGE gels before transferred to a PVDF membrane (Millipore, Billerica, MA, U.S.A.). The samples were blocked with 5% nonfat dry milk and incubated with primary antibodies (Supplementary Table S1) overnight at 4°C. The blots were washed three times and incubated with a HRP-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ, U.S.A.). Finally, the blots were visualized via enzyme-linked chemiluminescence using the ECL kit (Pierce Biotechnology, Rockford, IL, U.S.A.) and quantified by using ImageJ software.
6
Western Blot Analysis of Signaling Pathways
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Protein lysates were prepared from tissue or purified cDCs using RIPA buffer. Proteins were separated with SDS-PAGE and transferred to Immobilon PVDF membrane (Millipore). Membranes were blocked for 1 h at room temperature in PBST containing 5% (w/v) milk, incubated overnight at 4°C with primary antibodies and subsequently with HRP-conjugated secondary antibody (Amersham Bioscience). Antibodies against non-phospho (active) β-catenin, PPARγ, phospho-(Ser473) Akt and total Akt were purchased from Cell Signaling Technologies; antibodies against Tubulin, GAPDH were purchased from Genetex. Blotted proteins were detected using Luminata Forte Western HRP substrate (Millipore) and exposed on to Hyperfilm photo film (Amersham).
7
Western Blot Analysis of Protein Expression
8
Western Blot Protein Analysis
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Nuclear or whole-cell proteins derived from each cell sample were fractionated by SDS-PAGE, blotted onto a nitrocellulose membrane (Whatman, WHA10402506). Membranes were blocked for 2 hours at room temperature in 5% milk/TBS-Tween 20 and were incubated overnight at 4 °C with the primary antibodies listed in Reagents and Antibodies in the main text and subsequently with HRP-conjugated secondary antibody (1:5,000; Amersham Bioscience). Films then were scanned, and the intensity of the bands was quantified using ImageJ Software v.1.37c (NIH).
9
Western Blotting and Immunohistochemistry Analyses
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Western blotting (WB) and immunohistochemistry (IHC) were performed as described 9 . For WB, anti‐SERPINH1 and anti‐β‐actin antibodies (1:1000) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). HRP‐conjugated secondary antibody was from Amersham Biosciences (Little Chalfont, UK). The blots were quantified using NIH Image 1.62 program. The protein level was normalized with β‐actin. For IHC, sections were incubated with anti‐SERPINH1 antibody (1:100). Image‐Pro plus 6.0 (MediaCybernetics Inc., SilverSpring, MD, USA) was used to analyse optical densitometry.
10
Western Blot Analysis of PERK, eIF2α, and Phospho-eIF2α
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Cells were washed with PBS and lysed in SDS-PAGE loading buffer (1% SDS, 62.5 mM Tris–HCl pH 6.8, 10% glycerol). Lysates were sonicated and loaded on Any-kD SDS-PAGE gels (BioRad). Proteins were transferred onto nitrocellulose and probed with primary antibodies diluted in Tris-buffered saline supplemented with 0.1% Tween 20 and 5% BSA. The following antibodies were used: PERK (D11A8) (1:1000), eIF2α (#9722; Cell Signaling technology) (1:1000), phospho-eIF2α (Ser51) (44728G; Invitrogen). An HRP-conjugated secondary antibody (Amersham) was employed to detect immune-reactive bands using enhanced chemiluminescence (SuperSignal, Thermo Scientific).
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