MAM was purchased from Midwestern Research Institute (Kansas City, MO, USA). Fluriso™ (Isoflurane, USP) was purchased from MWI Animal Health (Boise, ID, USA). Chloral hydrate, protease inhibitor cocktail (P8340) and anti-mouse IgG-HRP (A4416) was sourced from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor® 594 goat anti-rabbit IgG (A11012) and ProLong® Gold antifade reagent with DAPI (P36931) were purchased from Life Technologies (USA). Anti-Parvalbumin antibody (Rb pAb to PV; abll427), Anti-GAPDH antibody (Ms mAb to GAPDH; ab9484) and goat pAb to Rb IgG-HRP (ab6721) were purchased from Abeam (Cambridge, MA, USA). Laemmli sample buffer (#161-0737), 10× Tris/glycine/SDS buffer (#161-0732), Mini-Protean TGX Any kD gels (#456-9035) and nitrocellulose/filter paper sandwiches, 0.2μm (#162-0213) were purchased from Bio-Rad (California, USA). Pierce ECL western blotting substrate (#32106) and Restore western blot stripping buffer (#21059) were purchased from Thermo Fisher Scientific (USA). High-performance chemiluminescence film (Amersham hyperfilm ECL; 28906839) was purchased from GE Healthcare. All other chemicals and reagents were of either analytical or laboratory grade, and purchased from standard suppliers.
Any kd mini protean tgx gel
Manufactured by Bio-Rad
Sourced in United States
The Any kD Mini-PROTEAN TGX gel is a pre-cast polyacrylamide gel designed for protein electrophoresis. It is compatible with the Bio-Rad Mini-PROTEAN electrophoresis system and can be used to separate proteins of varying molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p pvdf membrane, by Merck Group (3 mentions)
Goat anti rabbit igg h l hrp conjugate, by Bio-Rad (3 mentions)
Iblot system, by Thermo Fisher Scientific (3 mentions)
Amersham ecl, by Cytiva (3 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Chloral hydrate, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Tris glycine sds running buffer, by Bio-Rad (2 mentions)
Bright glo luciferase assay system, by Promega (2 mentions)
Anti dykddddk flag tag antibody clone 1e6, by Fujifilm (2 mentions)
Anti β actin antibody clone ac 74, by Merck Group (2 mentions)
0.2 μm nitrocellulose membrane, by Bio-Rad (2 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Anti neomycin phosphotransferase 2 antibody, by Merck Group (2 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Sds running buffer, by Bio-Rad (2 mentions)
31 protocols using any kd mini protean tgx gel
1
Immunoblotting Reagents and Protocols
2
Western Blot Analysis of Immune Signaling
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Cell lysates were prepared as previously described [21] (link) following stimulation as described above. Samples were separated using SDS-PAGE on Mini-protean TGX Any kD gels (Bio-rad) and transferred onto a nitrocellulose membrane using a wet transfer system. Membranes were blocked, washed, and proteins were analyzed by immunoblotting with standard methods using antibodies specific to IRF-7, IRF-3 (both from Cell Signaling Technology) and GAPDH (Abcam). Secondary antibodies conjugated to HRP were obtained from Jackson ImmunoResearch and immunoreactive bands were detected with the Immuno-Star HRP Substrate kit (Bio-Rad). For time courses, cells were stimulated as described above and lysed directly in Laemmli buffer (Bio-rad) and equal volumes were assayed as with other western blots using antibodies specific to STAT1 and pSTAT1Y701 (Abcam). Densitometry was performed using ImageJ (NIH), and proteins of interest were normalized to a reference protein (GAPDH).
3
Recombinant Spike Protein Detection
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Separated gradient fractions were applied to SDS-PAGE for subsequent staining, in-gel fluorescence, and immunoblotting. For SDS-PAGE, pre-cast Mini-PROTEAN TGX Any kD gels (Bio-Rad Laboratories, Hercules, USA) were used. Samples were prepared with reducing 6× Laemmli sample buffer and incubated for 5 min at 37 °C. SDS-PAGE gels were stained using InstantBlue Coomassie Protein Stain (abcam, Cambridge, UK). In gel fluorescence was recorded on a fluorescence scanner FujiFilm FLA-9000 (GE Healthcare, Chicago, USA) set to the EGFP channel. Immunoblotting onto Immobilon-P 0.45 µm PVDF membranes (Merck Millipore, Burlington, VT, USA) was carried out using the Power Blotter Semi-dry Transfer System (Invitrogen, Waltham, MA, USA). Membranes were blocked using 0.05% Tween20 and 5% skim dry milk powder in TBS and incubated for 30 min at 37 °C followed by 30 min at room temperature. After three washing steps with TBST, membranes were incubated overnight at 4 °C with mouse monoclonal Anti-His (C-term)/AP Ab (No. 46-0284, now R932-25) diluted at 1:3000. Following another two washing steps with TBST (15 min) and subsequent equilibration (5 min) with alkaline phosphatase buffer (100 mM Tris-Base, 100 mM NaCl, 5 mM MgCl2, pH 9.5), the recombinant 6×His-tagged spike protein was detected by incubation with BCIP/NBT Color Development Substrate (Promega, Madison, WI, USA).
4
Characterization of Cannabinoid Receptor Signaling
5
Quantifying Protein Expression in NSCLC Cells
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500,000 NSCLC cells were seeded in a 10-cm tissue culture plate and treated with different concentrations of SMI when the cell confluency was between 50–60%. After 48 hours, total cell lysate, subcellular fractions (cytosol/nuclear fractions) were prepared using NE-PER reagents as described above. The supernatant from total cell lysate, nuclear and cytosol fractions were quantitated by Bio-Rad protein assay (Bio-Rad, Hercules, California). Twenty micrograms of protein equivalent lysates were boiled in 4 x SDS sample loading buffer and run on mini-Protean TGX AnyKd gel (Bio-Rad). The proteins were transferred to nitrocellulose and Western blots were performed using rabbit monoclonal anti-PKM2 antibody (AbCam, Cambridge, MA) and incubated with IR Dye 800CW near-infrared fluorescent goat anti-rabbit IgG secondary antibody (LI-COR, Lincoln, NE). The membrane was washed three times and scanned using Odyssey CLx system (LI-COR, Lincoln, NE).
6
Western Blot Analysis of hMSCs
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hMSCs were lysed in radio‐immunoprecipitation assay (RIPA) buffer (New England Biolabs, Herts, UK, www.neb.com ) containing protease inhibitors (Roche Diagnostics, Burgess Hill, UK, www.roche.co.uk ). Total protein was measured using Bradford reagent (Sigma‐Aldrich, Dorset, UK, www.sigmaaldrich.com ). Protein supernatants were reduced in Lamelli buffer, separated on a Mini‐PROTEAN TGX Any kD gel and transferred to nitrocellulose membranes (all Bio‐Rad Laboratories, Hemel Hempstead, UK, www.bio‐rad.com ). Membranes were blocked in 5% milk for 60 minutes before overnight incubation with primary antibodies; rabbit polyclonal anti‐PPARγ (1:500; Santa Cruz Biotechnology, Heidelberg, Germany, www.scbt.com ), rabbit polyclonal anti‐IFT88 (1:500; Proteintech, Manchester, UK, www.ptglab.com ), and mouse monoclonal anti‐β actin (1:10,000 Abcam, Cambridge, UK, www.abcam.com ). Secondary antibodies used were IRDye 680RD goat anti‐mouse IgG (H+L) and IRDye 800CW donkey anti rabbit IgG (H+L) (both 1:15,000; LI‐COR Biotechnology, Cambridge, UK, www.licor.com/bio ). Membranes were imaged using an Odyssey infra‐red imaging system (LI‐COR Biotechnology, www.licor.com/bio ).
7
Analyzing GPx4 Protein Expression
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Two days after transfection with GPx4 siRNA or scrambled control siRNA, the proteins were extracted from cells and mouse retinas. SDS-PAGE of cellular proteins or retinal proteins was performed on Mini-PROTEAN TGX Any kD gel (Bio-Rad Laboratories, Hercules, CA) with Tris-glycine-SDS running buffer (Bio-Rad Laboratories). Immunoblot analysis was performed by electrotransfer of the proteins from the gels onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica MA) at 100 V for 60 min at ice-cold temperature using Tris-glycine buffer. The membranes were probed with antibodies for β-actin (Santa Cruz Biotechnology, Dallas, TX) and GPx4 (Cayman, Ann Arbor, MI). Binding of secondary antibodies, conjugated to alkaline phosphatase or horseradish peroxidase, was observed using a chemiluminescent substrate (Pierce, Waltham, MA).
8
Immunoblot Analysis of Antioxidant Enzymes
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For in vitro experiments, cells after 2 days of transfection with siRNA were used. For in vivo experiments, the dissected mouse corneas were used. Proteins were extracted from the cells and mouse corneas using LIPA buffer. As previously described 24 , SDS/PAGE of the proteins was performed on Mini‐PROTEAN TGX Any kD gel (Bio‐Rad Laboratories, Hercules, CA, USA) with tris‐glycine‐SDS running buffer (Bio‐Rad Laboratories). Immunoblot analysis was performed by electrotransferring proteins from the gels onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 100 V for 60 min at ice‐cold temperature using tris‐glycine buffer. The membranes were probed with antibodies to GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), catalase (Santa Cruz Biotechnology), GPx1 (Cell Signaling Technology, Danvers, MA, USA), GPx4 (Cayman, Ann Arbor, MI, USA), SOD1 (Santa Cruz Biotechnology), or SOD2 (GeneTex, Irvine, CA, USA). Binding of secondary antibodies, conjugated to alkaline phosphatase or to horseradish peroxidase, was visualized with 5‑bromo‐4‑chloroindol‐2‑yl phosphate/Nitro Blue tetrazolium substrate (Bio‐Rad Laboratories) or chemiluminescent substrate (Pierce, Rockford, IL, USA).
9
Western Blot Analysis of EcSSB Protein
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RDP317 cells containing the aforementioned mutants of interest from the bumping experiments were grown overnight in LB (supplemented with Amp, 50 μg ml−1 and Kan, 50 μg ml−1) at 37°C with shaking. To 100 μl of these saturated cultures was added 50 μl of 3× SDS PAGE loading buffer. The samples were boiled for 5 min then resolved on an Any kD Mini-PROTEAN TGX gel (Bio-Rad, Catalog #456-9036). Protein was then electrotransferred to a nylon membrane (Magna; Westboro, MA, USA; catalog No. N00HYB0010) and blocked with 5% (w:v) non-fat dry milk powder in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4). The membranes were then labeled with a 1:15 000 dilution (in TBST 5% non-fat dry milk) of rabbit antibodies raised against EcSSB, then a 1:15 000 dilution of anti-rabbit antibodies conjugated with horseradish peroxidase (GE Healthcare Life Sciences, Catalog No. NA934). The bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Catalog No. WBKLS0050).
10
Polyacrylamide Gel Electrophoresis of Oligos and LNPs
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