Protein samples were reduced with 100 mM DTT (final concentration) and heated at 95 °C for 5 minutes. For the analysis of large proteins (eIF4G1 and α-Fodrin), protein extracts were resolved on a NuPAGE® Novex 3%–8% Tris-Acetate gel (Invitrogen). For the analysis of other proteins, protein extracts were resolved on a Novex® 4%–20% Tris-Glycine Mini Gel (Invitrogen). After electrophoresis, proteins were transferred to 0.2 μm nitrocellulose membranes (Invitrogen) and incubated with the appropriate primary antibody (ESI Table 1† ). Membranes were blocked with BLOTTO [1× PBS containing 0.05% Tween-20, 5% nonfat dry milk from Bio-Rad (Blotting-Grade Blocker)] solution for 1 h and incubated with the primary antibodies in BLOTTO overnight at 4 °C. Membranes were washed three times (15 min, 10 min, 5 min) with 1× PBS containing 0.05% Tween-20 and incubated with a HRP-conjugated secondary antibody for 1 hour at room temperature. Membranes were washed three times (15 min, 10 min, 5 min) with 1× PBS containing 0.05% Tween-20, and bands were visualized with Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were exposed to BioMax MR double emulsion films (Eastman Kodak, Rochester, NY).
Novex 4 20 tris glycine mini gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Novex 4–20% Tris-Glycine Mini Gels are pre-cast polyacrylamide gels designed for protein separation and analysis. They feature a 4-20% gradient acrylamide concentration, which allows for the separation of a wide range of protein molecular weights on a single gel.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Mini gel tank, by Thermo Fisher Scientific (2 mentions)
Pierce power blotter stainer system, by Thermo Fisher Scientific (2 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Nupage novex 3 8 tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Blotting grade blocker, by Bio-Rad (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by LI COR (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Odyssey 9120 imager, by LI COR (1 mentions)
Intercept pbs blocking buffer, by LI COR (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Novex tris glycine sds sample buffer 2, by Thermo Fisher Scientific (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Benzonase, by Merck Group (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
16 protocols using novex 4 20 tris glycine mini gel
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Cellular Proteins
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HD1A and HD1B cells were lysed in the Cell Lytic M mammalian cell lysis/extraction buffer (Sigma-Aldrich) according to the manufacturer’s instruction. Protein samples were fractionated on a Novex 4–20% Tris-Glycine Mini Gel (Invitrogen). After transferring to the polyvinylidene difluoride membrane (Bio-Rad), the membrane was blotted with 5% nonfat dry milk in 1x PBS, and incubated with rabbit anti-p-S6 (Ser235/236), anti-S6, anti-p-Drp1 (Ser616), anti-Drp1, and anti-actin primary antibodies (from Cell Signaling), or rat anti-LAMP1, rabbit anti-Opa1 antibodies (from Santa Cruz). Following incubation with the secondary antibody that conjugated with horse radish peroxidase, proteins were visualized with chemiluminescent substrate (Thermo Scientific) under ChemiDox MP Image System.
3
Quantitative Analysis of Mbnl1 Protein
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Muscle tissue was homogenized by mortar and pestle in RIPA Lysis and Extraction Buffer (Thermo Scientific) with 1× Halt Protease Inhibitor Cocktail (Thermo Scientific) and 0.05 U/μl Benzonase (Sigma Aldrich) and protein concentration was quantified by Pierce BCA Protein Assay Kit (Thermo Scientific). Up to 30 μg protein was denatured in Novex Tris–glycine SDS sample buffer (2×) (Invitrogen) for 5 min at 85°C. NuPAGE Sample Reducing Agent (Invitrogen) was added to 1× final concentration prior to separating protein on Novex 4–20% Tris–glycine Mini Gel (Invitrogen) and transferring to nitrocellulose membrane (LI-COR) according to manufacturer's protocol. Following 1 h of blocking with Intercept (PBS) Blocking Buffer (LI-COR) at room temperature, membrane was incubated overnight at 4°C in blocking buffer with 0.2% Tween-20 and mouse monoclonal antibody to α-tubulin (clone DM1A, Abcam ab7291) at 1:10 000 dilution and rabbit polyclonal antibody to Mbnl1 (A2764) (8 (link)) at 1:10 000 dilution. Protein detection was performed using IRDye 680RD Goat anti-Rabbit IgG and IRDye 800CW Goat anti-Mouse IgG at 1:15 000 each with imaging on Odyssey 9120 imager (LI-COR). Band intensity quantification was performed using Image Studio Lite software (LI-COR).
4
Deglycosylation and Western Blot Analysis of HIV-1 gp120
5
Immunoprecipitation of Poly(ADP-ribose) Proteins
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At 48 h after transfection, HEK 293T cells were washed twice with PBS (phosphate-buffered saline) and lysed in IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Triton X-100) containing 1 μM ADP-HPD (Millipore) and cOmplete Mini EDTA-free inhibitor (Roche) for 20 min (with rotation at 4°C). The lysate was clarified by centrifugation at 4°C for 15 min at 18,800 ×g. A final concentration of 10 μg/mL cytochalasin B (Sigma) and 25 μM nocodazole (Sigma) were then added to the supernatant. Samples were then mixed with either anti-HA magnetic beads (Thermoscientific) or protein G magnetic beads (Thermoscientific) coupled with anti-pADPr (Abcam) (beads were pre-blocked with 1–2% BSA). After overnight incubation at 4°C, the beads were washed 4 times with IP buffer, re-suspended in SDS loading buffer, boiled for 5 min, electrophoresed on a Novex 4–20% Tris-Glycine mini-gel (Invitrogen), and then transferred onto PVDF membranes. Mouse anti-poly(ADP-ribose) polymer [10H] (1:1000; Abcam), mouse anti-HA [F-7] (1:3000; Santa Cruz), mouse anti-β-Actin [C4] (1:3000; Santa Cruz), and HRP-conjugated goat antibody to mouse (1:10,000; Azure Biosystems) were used for detection.
6
Western Blot Sample Preparation
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RKO cells at 70% confluency were scraped in PBS, pelleted, and snap-frozen, and then thawed in NP-40 lysis buffer containing Halt inhibitor cocktail. Samples were spun down, and the supernatants were transferred to Laemmli sample buffer and boiled. The samples were then run onto a Bolt 4–12% Bis-Tris Plus Gel (Thermofisher, NW04120BOX). H1299 cells at 70% confluence were scraped in NP-40 lysis buffer containing Halt inhibitor cocktail, and further lysed in Laemmli sample buffer. Samples were spun down, and the supernatants were boiled. The samples were then run onto a Novex 4–20% Tris-Glycine Mini Gel (ThermoFisher, XP04200BOX).
7
Extraction and Detection of Nuclear Proteins
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Nuclear proteins were extracted using EpiQuik Nuclear Extraction Kit II (EPIGENTEK) and were separated on a Novex 4–20% Tris-Glycine Mini Gel (Thermo Fisher) using XCell SureLock Mini-Cell Electrophoresis System (Thermo Fisher) and transferred to Hybond P 0.45 µm polyvinylidene fluoride membrane (GE Healthcare). After blocking in 5% skimmed milk, the membrane was incubated with primary antibodies (anti-SOX17, rabbit, monoclonal, Cell Signaling Technology, cat. no. 81778, dilution 1:1,000; anti-PRDM1, rabbit, monoclonal, Cell Signaling Technology, cat. no. 9115, dilution 1:500; anti-DMRT1, rabbit, monoclonal, Abcam, cat. no. ab126741, dilution 1:1,000; anti-LaminB1, rabbit, polyclonal, Abcam, cat. no. ab16048, dilution 1:1,000; Supplementary Table 6 ). The antibody binding was detected by horseradish-peroxidase-conjugated anti-rabbit IgG (Dako; dilution 1:2,000 in 0.01% TBST) in conjunction with the Western Detection System (GE Healthcare).
8
Western Blot Analysis of Argonaute-2 in Retinas
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Retinas from dim and PD mice were lysed in CellLytic™ Cell Lysis Buffer (Sigma-Aldrich, MO, USA) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). A total of 20 μg of protein was reduced and denatured then subjected to electrophoresis on a Novex™ 4–20% Tris-Glycine Mini Gel (Thermo Fisher Scientific). After separation, the protein was transferred to a nitrocellulose membrane (Bio-Rad, CA, USA), blocked in 3% BSA/PBS for 1 h, then incubated in primary anti-Argonaute-2 antibody (1:1000, ab32381, Abcam, Cambridge, UK) overnight at 4 °C. The following day, the membrane was incubated for 2 h in a HRP-conjugated Goat Anti-Rabbit IgG (H + L) secondary antibody (Bio-Rad) and developed with the ClarityTM Western ECL Substrate (Bio-Rad). After development, the membranes were imaged using the ChemiDoc™ MP Imaging System with Image Lab™ software (Bio-Rad). GAPDH (G9545, Sigma-Aldrich) was used a protein loading control.
9
Protein Expression Analysis of Retinal Damage
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Retinas from dim-reared (DR) mice (n=6) and mice exposed to 5 days of photo-oxidative damage (PD) (n=6) were lysed in CellLytic TM Cell Lysis Buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). A total of 20 µg of protein was denatured in LDS loading buffer (Thermo Fisher Scientific) and subjected to electrophoresis on a Novex™ 4-20% Tris-Glycine Mini Gel (Thermo Fisher Scientific). The protein bands were transferred to a nitrocellulose membrane (Bio-Rad, CA, USA), blocked in 3% BSA in PBS for 1 hours and then incubated with primary antibody (anti-AGO2 antibody
10
Western Blot Analysis of Cleaved Caspase-3
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Following treatment, cells were solubilized in radioimmunoprecipitation assay buffer (Sigma-Aldrich, R0278) and protein concentration in lysates were determined using Bradford reagent (Sigma-Aldrich, B6916). Equal amounts of protein were resolved by Novex™ 4-20% Tris-Glycine Mini Gels (Thermo Fisher Scientific, XP04200BOX) and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad, 1704159S). Membranes were blocked with Tris buffer saline (TBS) 0.1% Tween 20 buffer containing 5% BSA (Sigma-Aldrich, A7906) for one h. The blots were then incubated overnight at 4 ºC with anti-cleaved caspase-3 antibody (Cell Signaling Technology, 9664S) and β-tubulin (Cell Signaling Technology, clone D3U1W, 86298). This was followed by incubation with horseradish peroxidase (HRP) -labeled matching secondary antibodies. Immunoreactive bands were detected by Luminata™ HRP substrate (Millipore, WBLUR0500) and visualized using a MicroChemi imager (DNR Bioimaging Systems, Jerusalem, Israel).
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