Total protein content of the samples was assessed by a microplate protein assay (Bio-Rad, Hercules, CA), and equal amounts of protein per sample and known molecular weight markers were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred onto PVDF membranes (Whatman, Florham Park, NJ) and incubated for 1 h at room temperature with a blocking solution (3% bovine serum albumin) in Tris-buffered saline buffer containing 0.1% Tween 20 (TBST). The blocked membranes were incubated with primary antibodies for ERK, phospho-ERK, IRE1α and PDI (Cell Signaling), GRP94 (Enzo Life Science), GRP78 (BD Japan), phospho-eIF2α (Ser52) (Invitrogen), phospho-IRE1α (Ser724) (Novus Biological) and β-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C and washed three times with TBST. The membranes were incubated with a secondary antibody conjugated with horseradish peroxidase (GE Healthcare, UK) for 1 h at room temperature and washed. Immunodetection analyses were performed using a BM Chemiluminescence Blotting Substrate (POD) Kit (Roche Diagnostics, Mannheim, Germany).
Bm chemiluminescence blotting substrate pod kit
Manufactured by Roche
The BM Chemiluminescence Blotting Substrate (POD) Kit is a laboratory product designed for the detection of proteins using the chemiluminescence technique. It provides the necessary reagents for the visualization of protein bands on Western blots.
Automatically generated - may contain errors
Lab products found in correlation
Microplate protein assay, by Bio-Rad (1 mentions)
Secondary antibody conjugated with horseradish peroxidase, by GE Healthcare (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Phospho ire1α ser724, by Novus Biologicals (1 mentions)
Grp94, by Enzo Life Sciences (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Silverquest silver staining kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by GE Healthcare (1 mentions)
Fabp4, by Abcam (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
3 protocols using bm chemiluminescence blotting substrate pod kit
1
Western Blot Analysis of ER Stress Markers
2
Comparative Analysis of FABP4 and FABP5 Proteins
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Recombinant proteins of FABP4 (0.5 μg/lane) and FABP5 (0.3 μg/lane) and known molecular weight markers were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred onto PVDF membranes and incubated for 1 h at room temperature with a blocking solution (3% bovine serum albumin) in Tris-buffered saline buffer containing 0.1% Tween 20 (TBST). The blocked membranes were incubated with primary antibodies for FABP4 (Abcam, Cambridge, UK) or FABP5 (Hycult, Uden, Netherlands) overnight at 4°C and washed three times with TBST. The membranes were incubated with a secondary antibody conjugated with horseradish peroxidase (Cell Signaling, Danvers, MA) for 1 h at room temperature and washed. Immunodetection analyses were performed using a BM Chemiluminescence Blotting Substrate (POD) Kit (Roche Diagnostics, Mannheim, Germany). Silver staining of gels was also performed using a SilverQuest Silver Staining kit (Thermo Fisher Scientific, Yokohama, Japan).
3
Western Blot Protein Quantification
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Total protein content of the samples was assessed by a microplate protein assay based on Lowry's method, and equal amounts of protein per sample and known molecular weight markers were subjected to SDS‐PAGE. Proteins were electrophoretically transferred onto polyvinylidene fluoride membranes and incubated for 1 hour at room temperature with a blocking solution (3% BSA) in Tris‐buffered saline buffer containing 0.1% Tween 20. The blocked membranes were incubated with primary antibodies for FABP4 (Abcam), GAPDH (Santa Cruz Biotechnology), nitric oxide synthase 3 (NOS3; BD Biosciences), phosphorylated NOS3 (BD Biosciences), and actin overnight at 4°C and washed 3 times with Tris‐buffered saline buffer with 0.1% Tween 20. The membranes were incubated with a secondary antibody conjugated with horseradish peroxidase (GE Healthcare) for 1 hour at room temperature and washed. Immunodetection analyses were performed using a BM Chemiluminescence Blotting Substrate (POD) Kit (Roche Diagnostics). Densitometry was analyzed using ImageJ software.
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