Western blot analysis was performed on muscle homogenates as previously described [16 (link)]. Briefly, after electrophoretic separation on a 4–20% gradient acrylamide gel (Stain-free precast gel, Biorad, France) and electrotransfer to Immobilon P (Biorad, France), the membrane was incubated with primary antibodies and then HRP-labelled secondary antibodies (Jackson ImmunoResearch Laboratories). Signal quantification was performed using a ChemiDoc Touch apparatus (Biorad, France) and the Image Lab software (Biorad). The amount of the chosen protein in each sample was corrected for differences in loading using either the amount of myosin, GAPDH or the total amount of proteins using the stain free system from Biorad, and normalized to the amount of the same protein present in the control, set to 100% as described previously [15 (link)]. Ten human controls (muscle biopsy from individuals non-affected by neuromuscular disease) of different age have been used, from 3.5 to 64 years. For each sample (mouse and human), 2–3 Western blots have been performed, and the value for each sample corresponds to the mean ± SEM of the different Western blots.
Stain free precast gel
Manufactured by Bio-Rad
Sourced in France, United States
Stain-Free precast gels are a type of laboratory electrophoresis gel used for protein separation and visualization. These gels are pre-cast and ready-to-use, eliminating the need for manual gel casting. They utilize a proprietary stain-free technology that allows for direct visualization of proteins without the need for additional staining steps.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Hrp labelled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Chemidoc touch apparatus, by Bio-Rad (1 mentions)
Immobilon p, by Bio-Rad (1 mentions)
Stain free system, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Tgx stain free precast gel, by Bio-Rad (1 mentions)
Bm chemiluminescence western blotting substrate, by Roche (1 mentions)
Dm6000b, by Leica (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Precision plus protein unstained standard, by Bio-Rad (1 mentions)
Mini protean system, by Bio-Rad (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Gel doc ez system, by Bio-Rad (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Immobilon forte western hrp substrate, by Merck Group (1 mentions)
6 protocols using stain free precast gel
1
Quantitative Western Blot Analysis of Muscle Proteins
2
Tris-Glycine Stain-Free Protein Quantification
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Total protein bands were detected using Tris-Glycine eXtended Stain-Free technology. In brief, protein samples were loaded in a Tris-Glycine eXtended Stain-Free Precast Gel (Bio-Rad, Cat# 4568123). After electrophoresis, the total protein bands were imaged using a ChemiDoc MP imaging System (Bio-Rad). For the decellularized tissue samples, this method was also used to normalize the proteins of interest according to the total protein.
3
Western Blot Analysis of p53 in LV Myocardial Samples
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The Western blot analysis of p53 in LV myocardial samples was performed as described previously (Kollárová-Brázdová et al., 2021 (link)). Briefly, proteins in the LV myocardial samples were separated by SDS-PAGE using TGX Stain-Free precast gels (Bio-Rad, Hercules, CA). Immunodetection was performed with a mouse anti-p53 purified primary antibody (BP53-12; Exbio Praha a.s., Prague Czech Republic; dilution 1:1000) and an anti-mouse secondary antibody (P0447, Polyclonal Goat Anti-Mouse Immunoglobulin/HRP; DAKO Denmark A/S, Glostrup, Denmark; dilution 1:1000). A BM Chemiluminescence Western blotting Substrate (Roche) and Fusion Solo S imager coupled with a CCD camera (Vilber Lourmat GmbH, Eberhardzell, Germany) were used for signal detection. The results were normalized based on the total protein levels observed on the Stain-Free precast gels (Bio-Rad).
4
Localization and expression of tagged proteins
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Localization of tagged fluorescent proteins was analyzed in live cells grown in SC medium. Wide-field fluorescence images of GFP-tagged versions of ScPml39 or SpRsm1 were acquired using a Leica DM6000B microscope with a 100 ×/1.4 NA (HCX Plan-Apo) oil immersion objective and a CCD camera (CoolSNAP HQ; Photometrics), and further scaled equivalently using the MetaMorph software (Molecular Devices). Whole-cell extracts were prepared from cells grown in SC and analyzed by SDS-PAGE using stain-free precast gels (Biorad) followed by western-blotting with monoclonal anti-GFP antibodies (clones 7.1 and 13.1, Sigma)66 (link). Growth assays were achieved by spotting serial dilutions of cells on SC medium and incubating the plates at 25 °C.
5
Protein Quantification and Deglycosylation Protocol
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Protein quantification was performed at 280 nm with a Nanodrop 2000 (Thermo Fisher Scientific). The following parameters were used for quantification, ε280: 49 640/M/cm, molecular weight (MW): 29 506 Da for M-Fae and P-Fae, and MW: 30 760 Da for E-Fae. All parameters were estimated using ExPASy ProtParam tool (Gasteiger et al. 2005 ).
SDS-PAGE was performed with Mini-PROTEAN® system from Bio-Rad (Hercules, California, USA), Stain-Free™ Precast Gels, and Precision Plus Protein™ Unstained Standard, according to the manufacturer’s guidelines. Imaging was performed with a Gel Doc™ EZ system and Image Lab™ software (Bio-Rad). Protein molecular weights were estimated using the tools included in the Image Lab™ software. The enzymes were kept frozen (− 80 °C, 5% v/v glycerol) until use.
N-Glycans were removed using PNGaseF (New England Biolabs, Ipswich, Massachusetts, USA) under denaturing conditions, according to the supplier’s instructions and visualized by SDS-PAGE. Deglycosylation using non-denaturing conditions was also attempted according to the supplier’s instructions. No mobility shift was observed on SDS-PAGE after a first incubation at 37 °C for 16 h (data not shown), nor after addition of fresh PNGaseF and a second incubation at 37 °C for 4 h (data not shown).
SDS-PAGE was performed with Mini-PROTEAN® system from Bio-Rad (Hercules, California, USA), Stain-Free™ Precast Gels, and Precision Plus Protein™ Unstained Standard, according to the manufacturer’s guidelines. Imaging was performed with a Gel Doc™ EZ system and Image Lab™ software (Bio-Rad). Protein molecular weights were estimated using the tools included in the Image Lab™ software. The enzymes were kept frozen (− 80 °C, 5% v/v glycerol) until use.
N-Glycans were removed using PNGaseF (New England Biolabs, Ipswich, Massachusetts, USA) under denaturing conditions, according to the supplier’s instructions and visualized by SDS-PAGE. Deglycosylation using non-denaturing conditions was also attempted according to the supplier’s instructions. No mobility shift was observed on SDS-PAGE after a first incubation at 37 °C for 16 h (data not shown), nor after addition of fresh PNGaseF and a second incubation at 37 °C for 4 h (data not shown).
6
Protein Expression and Western Blot Analysis
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Cell lysates were subjected to electrophoresis on 4-15% gradient SDS-polyacrylamide gels (Stain free precast gels; Biorad). Proteins were transferred to PVDF membranes (Immobilon P, Millipore). Total proteins were visualized by stain free detection prior to transfer for comparison of sample loads, when necessary. After blocking in 5% nonfat dry milk or 3% BSA in TBS containing 0.05% Tween 20 (TBST) for 3 h at room temperature, membranes were incubated with primary antibodies overnight at 4 °C, in TBST containing either 2% nonfat dry milk or 1% BSA. After three washes of 10 min each in TBST, HRP-conjugated secondary antibodies were applied for 1 h. Membranes were extensively washed and signals resulting from the reaction with the Immobilon Forte Western HRP substrate (Millipore) were imaged with the ImageLab 6.0.1 software in a ChemiDoc imager (BioRad).
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