mes 20 × buffer  (Bio-Rad)

Journal: The Journal of Biological Chemistry

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1016/j.jbc.2025.110795

Figure Lengend Snippet: Materials

Article Snippet: MES 20× buffer , Bio-Rad , 1610789.

Techniques: Protease Inhibitor, Staining, Membrane

Journal: Bio-protocol

Article Title: Quantification of Folded and Misfolded Proinsulin Forms Using Nonreducing SDS-PAGE and Proinsulin-Specific Immunoblotting

doi: 10.21769/BioProtoc.5337

Figure Lengend Snippet: (1) Cells lysed in RIPA buffer with protease inhibitors on ice (15 min) and samples heated (95 °C, 5 min) in nonreducing or reducing gel buffer. (2) Separated on 12% NuPAGE gels (SDS-PAGE). (3) Nonreducing gels: one untreated and the other treated with 100 mM DTT (60 °C, 10 min, but can go up to 15 min). (4) Electrotransfer to nitrocellulose (semi-dry preferred, but wet transfer works), followed by 5% BSA blocking (room temperature) and three TBST washing steps (5 min each). (5) Nitrocellulose with primary proinsulin-specific antibody (1:1,000 in TBST + 5% BSA) incubated at 4 °C, overnight, followed by three TBST washing steps. (5) HRP-secondary antibodies (1:5,000) incubated at room temperature for 1 h, followed by three TBST washing steps. (6) Immunoblot developed with Bio-Rad Clarity Western ECL substrate; folded and misfolded proinsulin is quantified. The inset (bottom right) illustrates a representative immunoblot (cartoon) demonstrating the enhanced detection of proinsulin monomers, specifically native forms, in pancreatic β-cells using this modified nonreducing SDS-PAGE blotting method. This technique provides a more accurate quantification of proinsulin folding states. This modified immunoblotting protocol is based on Arunagiri et al. [7]. Protein Science , 33(4), e4949.

Article Snippet: NuPAGE TM MES SDS running buffer (20×) (Thermo Fisher, catalog number: NP000202) 13.

Techniques: SDS Page, Electrotransfer, Blocking Assay, Incubation, Western Blot, Modification

Journal: Bio-protocol

Article Title: Quantification of Folded and Misfolded Proinsulin Forms Using Nonreducing SDS-PAGE and Proinsulin-Specific Immunoblotting

doi: 10.21769/BioProtoc.5337

Figure Lengend Snippet: (A) Identical wells of MIN6 cells were treated ± Antimycin A (6 μM for 1.5 h), and equal aliquots of cell lysate or media (extracellular) were resolved by 12% NuPAGE under nonreducing (lanes 1–8) or reducing conditions (lanes 9–12). Identical nonreduced samples were either not exposed (lanes 1–4) or exposed (lanes 5–8) to post-gel disulfide reduction (as indicated below) prior to electrotransfer and immunoblotting with anti-rodent proinsulin (Proins). Non-native proinsulin monomers and disulfide-linked proinsulin dimers are indicated (red arrows); native proinsulin monomers migrate faster (blue arrow). (B) Quantitation of native proinsulin monomers recovered (lane 5 or 6, blue arrow) as a fraction of total proinsulin (lane 9 or 10) (n = 3 independent experiments; mean ± SD; *p = 0.0045). The quantification was done by band densitometry analysis using ImageJ. [Source: Arunagiri et al. [7], Protein Science , 33(4), e4949.]

Article Snippet: NuPAGE TM MES SDS running buffer (20×) (Thermo Fisher, catalog number: NP000202) 13.

Techniques: Electrotransfer, Western Blot, Quantitation Assay

Journal: Bio-protocol

Article Title: Quantification of Folded and Misfolded Proinsulin Forms Using Nonreducing SDS-PAGE and Proinsulin-Specific Immunoblotting

doi: 10.21769/BioProtoc.5337

Figure Lengend Snippet: Identical aliquots of MIN6 cell lysate and 6 h conditioned media were resolved by nonreducing 12% NuPAGE. After electrophoresis, the gel was cut longitudinally in four equal sections (dotted lines), and each quarter was treated as indicated (below the image) prior to electrotransfer to nitrocellulose for immunoblotting with anti-rodent proinsulin. Improved detection of secreted proinsulin (blue arrow, as well as intracellular proinsulin monomer bearing native disulfide bonds) is noted after post-gel disulfide reduction (compare lanes 6 or 8 with lanes 2 or 4). 15 min is the post-gel reduction time. [Source: Arunagiri et al. [7], Protein Science , 33(4), e4949.]

Article Snippet: NuPAGE TM MES SDS running buffer (20×) (Thermo Fisher, catalog number: NP000202) 13.

Techniques: Electrophoresis, Electrotransfer, Western Blot