Equal mass aliquots of NLP samples (1.5 μg) were diluted with 2x native gel sample buffer (Invitrogen) and loaded onto 4–12% gradient pre-made Tris-glycine gels (Invitrogen). Samples were electrophoresed for 2 hrs. at a constant 125 V. After electrophoresis, gels were incubated with SYPRO Ruby protein gel stain (Bio-Rad) for 2 hours and then de-stained using 10% Methanol, 7% Acetic acid. Following a brief wash with ddH2O, gels were imaged using the green laser (532 nm) of a Typhoon 9410 (GE Healthcare) with a 610 nm bandpass 30 filter. Molecular weights were determined by comparing migration vs. log molecular weight of standard proteins found in the NativeMark standard (Invitrogen).
Native gel sample buffer
Manufactured by Thermo Fisher Scientific
Native gel sample buffer is a solution used in the preparation of protein samples for native polyacrylamide gel electrophoresis (native PAGE). It is designed to maintain the native, non-denatured structure of proteins during sample loading and electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Sypro ruby protein gel stain, by Bio-Rad (2 mentions)
Typhoon 9410, by GE Healthcare (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
Nupage bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Mes sds buffer, by Thermo Fisher Scientific (1 mentions)
Dectin 1 antibody, by R&D Systems (1 mentions)
Biomax ms film, by Kodak (1 mentions)
Novex 4 20 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
5 protocols using native gel sample buffer
1
Native PAGE Analysis of NLP Samples
2
Native Gel Electrophoresis and Western Blot
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Equal amounts of NLP samples (1.25 μg) were diluted with 2x native gel sample buffer (Invitrogen) and loaded onto duplicate 4–20% gradient pre-made Tris-glycine gels (Invitrogen). Samples were electrophoresed for 140 min. at a constant 125 V. One gel was stained and scanned for total protein using SYPRO Ruby protein gel stain (Bio-Rad) as above. The second gel was transferred to a PVDF membrane (Invitrogen) for 100 V, 60 min. The native blot was blocked for 1 ½ hours in blotto (5% powdered milk in 1X PBS, Gibco), the blot was incubated with primary anti-YopD antibody (rabbit) in at a 1:500 dilution in blotto over night at 4°C with mixing. The blot was washed 3X for a total of 30 min. in 1X PBS with 0.05% tween-20. A secondary fluorescent antibody was used for detection in the following concentrations, rhodamine conjugated goat-anti-mouse IgG 1:500 in blotto (Upstate), for 3 hours at 4°C. The blot was washed as above and imaged using the green laser (532 nm) of a Typhoon 9410 (GE Healthcare) with a 555 nm band pass 30 filter.
3
Dectin-1 Immunoblotting: Optimized Protocols
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Immunoblotting for Dectin-1 was performed using an equal protein 30 μg of cell lysate or recombinant protein per lane. Samples were loaded on a 4%–12% precast NuPAGE Bis-Tris polyacrylamide gel (Invitrogen).55–58 (link) Proteins were electrophoresed using MES-SDS buffer at 200 V for 35–45 minutes per the manufacturer's instructions (Invitrogen). Native gel electrophoresis was performed using a Novex 4% Tris-glycine gel (Invitrogen). Samples were mixed with a cold native gel sample buffer (Invitrogen) before loading. Electrophoresis was run at room temperature at a constant voltage of 80 V for 5 hours. Proteins were then transferred from the gel to a nitrocellulose membrane using the XCell II Mini-Cell and sandwich blot module (Invitrogen) in Tricine-10% methanol-0.01% SDS transfer buffer (Invitrogen) at 30 V for 1 hour. Blots were blocked for 1 hour at room temperature with 10% nonfat milk and then incubated with primary Dectin-1 antibody (R&D system) (1:500 in 1% nonfat milk) overnight at 4°C. Blots were then incubated with 1:5000 goat anti-rabbit IgG-horseradish peroxidase for 2 hours at room temperature. Signal was detected using the enhanced chemiluminescence kit (Amersham), and blots were exposed to Kodak Biomax MS film.
4
Protein Visualization by SDS-PAGE
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Elution fractions from size-exclusion chromatography were mixed with native gel sample buffer (Invitrogen) and loaded on a Novex 4–20% Tris-Glycine gel (Invitrogen). Staining was carried out with InstantBlue Protein Stain (VWR).
5
STING Protein-Ligand Binding Assay
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Purified hSTING protein at 20 µM in buffer B was incubated with the compounds (cGAMP, c53, NVS-STG2 or their different combinations) at 40 µM at 4 °C overnight. The samples were mixed with the native gel sample buffer (Invitrogen, cat. no. BN20032) and resolved by a 3–12% gradient native gel (Invitrogen, cat. no. BN2012BX10).
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