The turnover of ΔssCPY∗-GFP and Hmg2-myc was assessed in cells by inhibiting protein synthesis with cycloheximide. Cells were collected at the indicated time points and ΔssCPY∗-GFP and Hmg2-myc levels analyzed by immunoblotting and quantified by densitometry. Values shown are percentages relative to zero time points from three independent biological repeats. Protein extracts were electrophoresed under reducing conditions on NuPAGE minigels (Thermo Fisher Scientific) and electroblotted onto polyvinylidene fluoride membrane (GE Healthcare). Primary antibodies used were rabbit α-Sup35 (Ness et al., 2002 (link)), ubiquitin (sc-8017; Santa Cruz Biotechnology, Inc.), Ydj1 (ab74442; Abcam), Sis1 (COP-080051; Operon Biotechnologies), Ssa1 (ADI-SPA-822; Enzo Life Sciences), GFP (A6465, Invitrogen), Pgk1 (459250; Thermo Fisher Scientific), Hsp104 (ab2924; Abcam), rabbit α-Sse1 (Chiabudini et al., 2012 (link)), and Myc 4A6 (05–724; EMD Millipore).
Nupage minigels
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE minigels are precast polyacrylamide gels designed for protein electrophoresis. They provide consistent, high-resolution separation of proteins under denaturing conditions.
Automatically generated - may contain errors
Lab products found in correlation
Ab2924, by Abcam (1 mentions)
Ubiquitin sc 8017, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by GE Healthcare (1 mentions)
Myc 4a6, by Merck Group (1 mentions)
Nupage mops, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Typhoon scanner, by Cytiva (1 mentions)
Brca1 a8x9f, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
5 protocols using nupage minigels
1
Protein Turnover Dynamics Analysis
2
Proteomic Analysis Using Protease Inhibitors
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Complete protease inhibitor cocktail tablets (Cat No: 5892970001) were obtained from Roche. Bicinchoninic Acid Assay (BCA) Kit (Cat No: 23227) was from Pierce. InstantBlue staining kit was obtained from Expedeon (Cat No: ISB). NuPAGE minigels (Cat No: NP0321BOX), LDS sample buffer (Cat No: NP0007) and NuPAGE MOPS (Cat No: NP0001) and MES running buffers (Cat No: NP0002) were from Thermo Fisher. Trypsin was obtained from Promega (Cat No: 90058). C18 cleaning columns were from Applied Biosystems (Cat No: 1112906) and the Pepmap C18 columns were from Dionex (Cat No: 160321). All other reagents were obtained from Sigma.
3
Western Blot Analysis of Muscle Proteins
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The C2C12 controls on TCP and PLCL scaffolds were lysed with RIPA buffer at T0 and from T1 to T10 as described by Costa et al. [35 (link)]; then, lysates were collected and stored at −80 °C. Individual protein concentration measurements were performed using a Lowry assay kit. Proteins were separated on Invitrogen NuPage mini gels (Thermo Fisher Scientific, Waltham, MA, USA), transferred to a nitrocellulose membrane and blocked in 5% skimmed milk dissolved in TBS-Tween (0.1% Tween) for 1 h at RT. Membranes were incubated overnight at 4 °C with primary antibody antiskeletal muscle myosin (F59) (dilution 1:200; Santa Cruz Biotechnology, Dallas, TX, USA) and incubated for 1 h at RT with peroxidase-labeled secondary antibody. After washing, membranes were exposed for 1 minute to ECL Western blotting substrates (1:1), and chemiluminescent signals were detected (Clarity Western ECL Substrate, BioRad Hercules, CA, USA). The relative intensity of protein expression was calculated using ImageJ2 and normalized to actin.
4
Monitoring OMP Folding and Assembly
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Aliquots of assembly reactions were mixed with SDS-PAGE sample buffer and either heated at 95 °C for 5 min or left unheated before proteins were resolved on 8–16% NuPAGE minigels (Thermo Fisher Scientific). In general, assembly was assessed by comparing heated and unheated samples and monitoring the appearance of a fast migrating species in the absence of heat that corresponds to the folded form of the OMP. The assembly of EspP∆5 was also analyzed by monitoring the cleavage of the protein (and the appearance of a free β barrel domain fragment) in an autocatalytic reaction that requires the protein to fold into a native conformation. To separate the folded and unfolded forms of OmpG, SDS-PAGE was conducted at 4 ° C. Fluorescently-labeled in vitro translated OMPs were visualized using an Amersham Typhoon scanner at an excitation wavelength of 488 nm. The folded fraction was quantitated using ImageJ software and plotted or fit to exponentials using Igor Pro as described30 (link). In experiments in which the assembly of urea-denatured OMPs was analyzed, proteins were detected by Western blot as previously described30 (link).
5
Protein Analysis by SDS-PAGE and Western Blot
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Protein fractions were analyzed by SDS-PAGE followed by either Coomassie blue staining or Western blotting. Proteins were separated on 3 to 8% tris-acetate NuPAGE mini gels (Thermo Fisher Scientific) and stained by SimplyBlue SafeStain solutions (Invitrogen) for 60 min. Gels were washed with deionized water for 60 min and then 3% NaCl for an additional 2 hours—overnight to achieve maximum sensitivity. Western blots were performed as described previously (16 (link)). The following primary antibodies were used in our analysis: BRCA1-C20 (SCBT, sc-642; α-BRCT), BRCA1-Ab1 (Calbiochem, OP92; α-RING), BRCA1-A8X9F (Cell Signaling Technology, #14823; α-RING), BARD1 (SCBT, sc-11438), ubiquitin-pAb (Enzo Life Sciences, ADI-SPA-200), RAD51 (SCBT, sc-8349), and β-actin (Sigma-Aldrich, A5441).
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