Cells were washed with ice-cold PBS and scraped into ice-cold lysis buffer with phosphatase inhibitor cocktail (Stratech, B15001-BIT). Lysates were cleared by centrifugation at 14 000 rpm for 10 min at 4 °C, and supernatants were collected and assayed for protein quantification using the Bradford protein assay (Biorad, 5000006). Quantified samples were then analyzed by western blotting. Twenty micrograms of total lysate were resolved on NuPAGE 4–12% Bis-Tris gels and electrophoretically transferred to PVDF membranes (Biorad, 1704157). Membranes were blocked for 1 h in 5% BSA in Tris-buffered saline (TBS)-Tween and then hybridized using the primary antibodies in 5% BSA TBS-Tween. Mouse and rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20 000, GE Healthcare) were diluted in 5% BSA in TBS-Tween. Protein-antibody complexes were detected by chemiluminescence with ECL λernova (Cyanagen) and images were captured with a c300 azure camera system. Protein bands were analyzed by comparing their size to SMOBIO-PM-2600 protein marker.
Nupage 4 12 bis tris gels
Manufactured by Bio-Rad
Sourced in United States
The NuPAGE 4–12% Bis-Tris gels are pre-cast polyacrylamide gels designed for protein separation and analysis. They feature a Bis-Tris buffer system and a gradient of 4% to 12% acrylamide concentration, providing effective resolution of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Bradford protein assay, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Goat anti rabbit ig hrp, by Agilent Technologies (1 mentions)
Complete tablet, by Roche (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Rabbit anti mouse ig hrp secondary antibody, by Agilent Technologies (1 mentions)
Primary antibodies against gapdh, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Cldn1, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti loricrin, by BioLegend (1 mentions)
Keratin 10, by BioLegend (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
2 d quant kit, by GE Healthcare (1 mentions)
5 protocols using nupage 4 12 bis tris gels
1
Western Blot Analysis of Protein Lysates
2
Protein Expression Analysis in Lung Tissue
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Frozen lung tissues were homogenized in RIPA buffer (Sigma) containing protease inhibitors (Complete tablets, Roche, Indianapolis, IN, USA). Samples were equalized according to the protein concentration, boiled for 1 min in 4x loading buffer, separated by SDS-PAGE on NuPage 4–12% Bis-Tris gels (Bio-rad, Hercules, CA, USA) and transferred onto polyvinylidene fluoride membranes using the semidry transfer system (Bio-rad). The membranes were then blocked for 2 h at room temperature with 5% nonfat dry milk (Bio-rad) in Tris-buffered containing 0.5 M NaCl and 0.1% Tween (TBST), incubated with primary antibodies against GAPDH (1:500, Santa Cruz) or LC3B (1:5000, Sigma) overnight at 4°C, washed with TBST and incubated with goat anti-rabbit Ig-HRP or rabbit anti-mouse Ig-HRP secondary antibody (1:5000 and 1:10000, respectively, both from Dako, Glostrup, Denmark). Protein expression was detected using ECL reagents and quantified by densitometry using Image Studio Lite software (LI-COR Biotechnology).
3
Monitoring Epithelial Differentiation Markers
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N/TERT2G cells were plated in 24-well plates at 75,000 cells/well. Cell lysates (RIPA buffer containing protease and phosphatase inhibitors with 0.2% SDS) were collected from undifferentiated cells and from cells on days 1, 2, and 3 after differentiation. Samples were run on Invitrogen NuPAGE 4‒12% Bis-Tris gels and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were probed with the antibodies provided in Table 4 . Antibodies (Table 4 ) were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). Relative protein expression was determined by densitometry calculated using ImageJ software (National Institutes of Health, Bethesda, MD). Samples were normalized to β-actin expression and protein content (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific).
Western Blot Antibodies
| Antibodies | Company | Dilution |
|---|---|---|
| Anti-CLDN1 (519000) | Invitrogen | 1:1,000 |
| Anti‒β-actin (C4) HRP | Santa Cruz | 1:5,000 |
| Anti‒keratin 10 (Poly 19054) | BioLegend | 1:1,000 |
| Anti-loricrin (Poly 19051) | BioLegend | 1:1,000 |
| Anti-occludin (OC3F10) | Invitrogen | 1:500 |
| Anti-mouse IgG HRP (NA931V) | Sigma-Aldrich | 1:5,000 |
| Anti-rabbit IgG HRP (NA934V) | Sigma-Aldrich | 1:5,000 |
Abbreviation: HRP, horseradish peroxidase.
4
Immunoblot Protocol for Protein Analysis
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Immunoblot was performed as follows: cells or grinded tissues were homogenized in NP40 buffer, debris were removed by centrifugation (20 000 g, 10 min, 4°C). Protein content in the supernatant was measured using the Bradford assay (Biorad) and samples were separated on NuPage bis-tris 4–12% gels in MOPS buffer. Separated proteins were transferred to nitrocellulose membranes (Biorad). Membranes were blocked with phosphate-buffered saline-Tween (0.05%) and milk (5%). Primary antibodies were incubated overnight. Antibody Rb11 was generated by immunization of rabbits with a truncated FMRP protein corresponding to its C-terminal region, as previously described (10 (link)). Rb11 was used at the dilution of 1:500 for Western blot. Monoclonal 1C3 anti-FMRP antibody (24 (link)) was used at the dilution 1:1000; mouse polyclonal anti-FMRP 1R was used at the dilution 1:1000 (11 (link)). Anti PDE2A (Fabgennix, # PD2A-101AP), PSD95 (Millipore, AB9708), Anti-rpS6 (Cell signaling Technology, 54D2) anti ß-Actin (Sigma, clone AC-74).
5
Protein Quantification and Immunoblotting Protocol
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