500 nmol of 6×His-tagged Get4-5N was incubated with 10 µL Ni-NTA agarose resin for one hour at 4°C in 500µL binding buffer containing 20 mM K-HEPES pH 7.5, 150 mM KOAc, 10 mM MgOAc, 10% (v/v) Glycerol, 25 mM imidazole, and 1 mM ADP or ATP where indicated. Following the addition of 1 µmol of Get3D, the solution was incubated for an hour at 4°C. After incubation, the reaction was spun for 30sec at 500×g. The supernatant was removed, and 500 µL binding buffer was added to the solution, and gently mixed through inversion. The wash step was repeated twice, and following the final wash, the remaining bound proteins were eluted with 30 µL of 20mM Tris pH 7.5, 100 mM NaCl, 5mM BME, and 300 mM imidazole. The samples were spun for 30sec at 500 × g, and the supernatant was removed and added to 6 µL of 6×SDS-PAGE buffer. All samples were run on 15% SDS-PAGE gels and stained with Coomasie blue G-250. Gels were then analyzed by infared scanning in the 700 nm channel using a LI-COR Odyssey Infared Imaging System and Odyssey Application Software v3.0.30.
Odyssey infared imaging system
Manufactured by LI COR
Sourced in United States
The Odyssey Infrared Imaging System is a laboratory instrument designed for high-sensitivity fluorescence detection and quantitative analysis of proteins and other biomolecules. It utilizes near-infrared fluorescent dyes and provides a range of features for researchers to accurately and efficiently conduct their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey application software v3, by LI COR (2 mentions)
Actin, by Santa Cruz Biotechnology (1 mentions)
Pi3 kinase p110β, by Cell Signaling Technology (1 mentions)
Stat5, by Cell Signaling Technology (1 mentions)
Pi3 kinase p110α, by Cell Signaling Technology (1 mentions)
Akt antibody, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Intercept tbs blocking buffer, by LI COR (1 mentions)
Revert 700 total protein stain, by LI COR (1 mentions)
Anti foxm1, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene di uoride membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
7 protocols using odyssey infared imaging system
1
Affinity Purification of Get3-Get4/5 Complex
2
Western Blotting of PI3K Pathway Proteins
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GC tissues or GC cells were lyzed in RIPA buffer for 30 min on ice supplemented with a protease inhibitor cocktail, followed by centrifugation at 12,000 rpm for 10 min. The lysates was then diluted in 5 × SDS loading buffer and boiled for 3 min. The protein mixtures were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro‐blotted to a PVDF membrane (EMD Millipore). After being blocked with non‐fat milk for about 1 h at room temperature, the membrane was then incubated 2 h with the primary antibodies at room temperature or overnight at 4°C, followed by the secondary antibody for another 1 h. The protein detection was performed with Odyssey Infared Imaging System (Li‐COR). Primary antibodies used in this study as follows: actin (1:500, Santa Cruz Biotechnology, #8432), PI3 Kinase p110α (1:1000, Cell Signaling Technology, #4249), PI3 Kinase p110 δ (1:1000, Cell Signaling Technology, #34050), PI3 Kinase p110β (1:1000, Cell Signaling Technology, #3011), STAT5 (1:1000, Cell Signaling Technology, #25656), JAK3 (1:1000, Cell Signaling Technology, #8827), Phospho‐Akt (Ser473) (1:1000, Cell Signaling Technology, #9271), Akt Antibody (1:1000, Cell Signaling Technology, #9272).
3
Western Blot Analysis Protocol
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SDS PAGE gels (10%) and PVDF membranes were used. Membranes were blocked using Intercept (TBS) Blocking Buffer (Licor), total protein quantification was performed by using Revert 700 Total Protein Stain (Licor), and primary antibody overnight incubation was performed at 4°C, followed by 30-min IRDye (Licor) secondary antibody incubation at room temperature. Blots were imaged using Licor Odyssey Infared Imaging system.
4
Western Blot Analysis of GC Cell Lysates
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Cell lysates from GC cells were prepared using cold lysis buffer (25 mmol/L Tris-Cl [pH7.5], 1% sodium dodecylsulfate [SDS], 5mmol/L ethylenediaminetetraacetic acid, protease inhibitor cocktail [Sigma-Aldrich, St Louis, USA]). Samples were boiled for 3 min, separated by electrophoresis in SDS-PAGE (10% w/v) and transferred to a nitrocellulose membranes. The membrane was blocked in 5% blocking buffer (5% non-fat milk and 0.1% Tween-20 in phosphate-buffered saline [PBS]) for 2 h at room temperature, and then incubated with the primary antibody, anti-PARI (1:500, ABGENT, USA), anti-FOXM1 (1:200, Santa Cruz Biotechnology, USA) and anti-GAPDH (1:20000, Proteintech, China), in 0.1% Tween-20 in PBS overnight at 4°C. Incubation with the secondary antibody was performed for 1 h at room temperature. Detection of proteins was achieved by using the Odyssey Infared Imaging System (Li-COR, USA).
5
Affinity Purification of Get3-Get4/5 Complex
6
Quantifying Protein Levels via Infrared Imaging
7
Carbonic Anhydrase II Expression in CCA
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CCA cells were seeded in a 6-well plate at a density of 1.2×10 6 cells/well. After growing to 80% of con uence, cells were treated either with Met (0, 10, 20, and 50 mM) for 24 h, or 50 mM Met for 0 h, 48 h, and 72 h. Total proteins from CCA cells were extracted in RIPA buffer. BCA protein assay kit (Takara Bio Inc., Otsu, Shiga, Japan) was used to measure the protein concentrations. Cell lysates were electrophoresed by SDS-PAGE, and the protein was then transferred onto polyvinylidene di uoride membranes (EMD Millipore Corp., Kenilworth, NJ, USA). The membranes were blocked by 5% non-fat milk for 1 h at RT, followed by incubation with primary antibody anti-CA2 (1:1000, Cat. PB1045, Boster Biological Technology, Wuhan, China) overnight at 4°C. After three washes with PBST for 5 minutes each, membranes were probed with HRP conjugated secondary antibody (1:2000, SC-2004, Santa Cruz Biotechnology, Dallas, TX, USA), then hybridization bands were visualized in the Odyssey Infared Imaging System (Li-COR, USA). β-actin (Santa Cruz Biotechnology, USA) served as the internal control.
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