A total of 0.08 million MIA PaCa-2 cells were seeded per well in a 24-well poly-d -lysine-coated cell culture plate (Greiner) and allowed to attach overnight before mRNA transfection with Lipofectamine™ MessengerMAX™ (Life Technologies) according to the manufacturer's protocol. After 24 h incubation, 100 μl of Bolt™ lithium dodecyl sulfate (LDS) sample buffer supplemented with Bolt™ sample reducing agent was added per well of a 24-well plate. The wells were scraped using wide orifice tips and the lysate was transferred into polymerase chain reaction (PCR)-strip tubes and sonicated for 10 × 10 s in a chilled water bath sonicator (QSonica). A 15 μl aliquot of protein extract was separated on 4–12% Bis-Tris plus gels, transferred onto nitrocellulose membranes using the Trans-Blot® Turbo™ semi-dry system (Bio-rad), and blocked for 1 h at room temperature with Intercept™ (TBS) blocking buffer (Li-Cor). Blots were probed with the appropriate primary antibodies overnight at 4°C in blocking buffer supplemented with 0.1% Tween-20, followed by the secondary antibodies IRDye® 680RD donkey anti-mouse IgG or IRDye® 800CW donkey anti-rabbit IgG (Li-Cor) for 1 h at room temperature. Fluorescent signals were imaged and quantified using Odyssey® CLx. Primary antibodies used were: NanoLuc (Promega, N7000) and glyceraldehyde phosphate dehydrogenase (GAPDH; Cell Signaling Technology, #5174)
Trans blot turbo semi dry system
Manufactured by Bio-Rad
Sourced in Italy
The Trans-Blot® Turbo™ semi-dry system is a laboratory equipment designed for efficient protein transfer from polyacrylamide gels to membranes. It utilizes a semi-dry electroblotting method to facilitate the rapid and consistent transfer of proteins for various downstream applications, such as Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 680rd donkey anti mouse igg, by LI COR (2 mentions)
Irdye 800cw donkey anti rabbit igg, by LI COR (2 mentions)
Poly d lysine coated cell culture plate, by Greiner (2 mentions)
Lipofectamine messengermax, by Thermo Fisher Scientific (2 mentions)
Nanoluc, by Promega (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Water bath sonicator, by Qsonica (2 mentions)
Intercept tbs blocking buffer, by LI COR (2 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Hif 1α, by BD (1 mentions)
C rel, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Calnexin, by Cell Signaling Technology (1 mentions)
Precision plus protein dual colour standard, by Bio-Rad (1 mentions)
Mini protean tgx 4 20 gel, by Bio-Rad (1 mentions)
Ab180851, by Abcam (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
6 protocols using trans blot turbo semi dry system
1
Quantifying NanoLuc protein expression
2
Protein Quantification and Immunoblotting Protocol
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Protein quantity was measured with Bradford reagent (BioRad) and prepared in SDS loading dye. For detecting HIF1α, a sample of the cells was harvested in 8 M Urea, sonicated and the protein quantified with the BCA assay (ThermoFisher). Between 5 and 10 µg of protein per well was loaded on SDS/PAGE (BioRad), separated and transferred with the Trans Blot Turbo semi dry system (BioRad) to PVDF (Millipore) membranes. The following antibodies were used for the present study at a dilution 1:1000: RelA C-terminal (sc-372, SantaCruz); RelA N-terminal (#4764, Cell Signalling); IκBα (#9242, Cell Signalling); p105/p50 (#3035, Cell Signalling); p100/p52 (#4882, Cell Signalling); RelB (#4954, Cell Signalling); c-Rel (#4727, Cell Signalling); α-tubulin (#2144, Cell Signalling); VDAC (#4866, Cell Signalling); protein disulphide isomerase (PDI) (sc-20132, Santa Cruz); TOM40 (sc-11414, Cell Signalling); AIF (sc-13116, Cell Signalling); TIM50 (SBS0666, Source BioSciense); Mortalin (MA3-028; Thermo Fisher); calnexin (#2433, Cell Signalling); HIF1α (610958, BD Transduction Laboratories); STAT3 (#9139, Cell Signalling).
3
Quantifying NanoLuc protein expression
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A total of 0.08 million MIA PaCa-2 cells were seeded per well in a 24-well poly-d -lysine-coated cell culture plate (Greiner) and allowed to attach overnight before mRNA transfection with Lipofectamine™ MessengerMAX™ (Life Technologies) according to the manufacturer's protocol. After 24 h incubation, 100 μl of Bolt™ lithium dodecyl sulfate (LDS) sample buffer supplemented with Bolt™ sample reducing agent was added per well of a 24-well plate. The wells were scraped using wide orifice tips and the lysate was transferred into polymerase chain reaction (PCR)-strip tubes and sonicated for 10 × 10 s in a chilled water bath sonicator (QSonica). A 15 μl aliquot of protein extract was separated on 4–12% Bis-Tris plus gels, transferred onto nitrocellulose membranes using the Trans-Blot® Turbo™ semi-dry system (Bio-rad), and blocked for 1 h at room temperature with Intercept™ (TBS) blocking buffer (Li-Cor). Blots were probed with the appropriate primary antibodies overnight at 4°C in blocking buffer supplemented with 0.1% Tween-20, followed by the secondary antibodies IRDye® 680RD donkey anti-mouse IgG or IRDye® 800CW donkey anti-rabbit IgG (Li-Cor) for 1 h at room temperature. Fluorescent signals were imaged and quantified using Odyssey® CLx. Primary antibodies used were: NanoLuc (Promega, N7000) and glyceraldehyde phosphate dehydrogenase (GAPDH; Cell Signaling Technology, #5174)
4
Colonic Mucus Immunoblotting Protocol
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Mucus was collected from mouse or human colonic biopsies after removal of luminal content, by gentle scraping and was collected in Kreb's buffer containing 2× cOmpl and 250 μM N-Ethylmaleimide. Samples for gel electrophoresis and western blot analysis were reduced in DTT and run in Mini-PROTEAN TGX 4–20% gels (Bio-Rad) with SDS-containing running buffer. The proteins were blotted to PVDF membranes using a semi-dry trans-blot turbo system (Bio-Rad) with the appropriate buffers. The membrane was probed with a rabbit monoclonal anti-CLCA1 primary antibody (ab180851, Abcam, RRID:AB_2722611 ) at 1:10000, and a goat anti-rabbit alkaline phosphatase conjugated secondary antibody (Southern Biotech, RRID:AB_2722612 ) at 1:1000. Nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate (NBT/BCIP) was used for development. Precision Plus Protein™ Dual Colour Standard (Bio-Rad) was used as molecular weight standard.
5
Western Blot Protein Expression Analysis
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Cells were lysed on ice using RIPA Lysis buffer kit with protease inhibitor cocktail (Santa Cruz Biotechnology Inc., Heidelberg, Germany). Protein concentration was measured using a BCA protein assay kit (Pierce, Thermo Scientific, Rockford, U.S.A). Thirty μg of reduced proteins in Laemmli sample buffer (Biorad, Segrate, Milan, Italy) were resolved using Gel precast Miniprotean 4-20% (Biorad) and transferred to nitrocellulose by Semi-Dry Trans-Blot Turbo System (Biorad). Membranes were blocked with 5% non-fat dry milk (Biorad) in TBS-T, and incubated with primary antibody overnight at 4°C and thereafter with the appropriate HRP-conjugated secondary antibody (Biorad) for 1h at room temperature. Membranes were developed using ECL detection reagent (Amersham, Glattbrugg, Switzerland). The primary antibodies were: anti-EP-CAM, anti-E-cadherin, anti-Vimentin (1:1000) and anti-α-Tubulin (1:2000) (Cell Signalling Technology, Massachusetts, USA). Blots were stripped for 20 minutes using the stripping buffer (Thermo Scientific) before re-probing.
6
Protein Extraction and Immunoblotting of NAc
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Mice were sacrificed by cervical dislocation, brains were removed and dissected on ice to isolate the NAc, then flash-frozen on dry ice before transferring to -80 °C for long-term storage. The day of the experiment, the tissue was weighed and homogenised on ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% triton X-100, pH 8.0) with phosphatase inhibitors (1 mM NaF and 0.1 mM Na3VO4) and protease inhibitor cocktail (Calbiochem, catalogue# 539134-1SET, 1:100). Extracts were rocked at 4 °C for 20 min and centrifuged at 10,000 x g for 20 min at 4 °C to isolate protein. Protein was quantified using BioRad DC Protein assay (BioRad, Catalogue# 5000112). 25 μg of protein were loaded onto 4–12% Bis-Tris Plus Gels (ThermoFisher) and protein was transferred onto PVDF membranes by BioRad Semi-Dry Trans-Blot Turbo System. The primary antibodies used for immunoblotting were: rabbit anti-VAChT (Synaptic System, catalogue# 139103, 1:2000), rabbit anti-synaptophysin (Cell Signaling Technology, catalogue# 5461S, 1:3000), rabbit anti-mCherry (Abcam, ab167453, 1:2000) and mouse anti-β-actin (Sigma-Aldrich, catalogue# A3854, 1:25000) as loading control. The secondary antibody was goat anti-rabbit HRP (BioRad, Catalogue# 170-6515, 1:7500). Proteins were visualised using chemiluminescence with the ChemiDoc MP Imaging System (BioRad).
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