Cryopreserved colon was homogenized using the BeadBug Microtube Homogenizer and 1.5 mm Triple-Pure High Impact Zirconium pre-filled tubes (Benchmark Scientific, Sayreville, NJ) with T-PER tissue protein extraction reagent (ThermoFisher Scientific) and protease inhibitor cocktail (ThermoFisher Scientific). Colon proteins were quantified using the Pierce BCA Protein Assay Kit per manufacturer’s protocol (ThermoFisher Scientific), and analyzed using Proteome Profiler Mouse Cytokine Array Kits (R & D Systems, Minneapolis, MN) per manufacturer’s protocol. Twentyfive μL of serum per mouse (N=4) was pooled by treatment. Membranes were imaged on a LAS-3000 luminescent imager (Fujifilm, Tokyo, Japan) and analyzed using ImageJ software.
Las 3000 luminescent imager
Manufactured by Fujifilm
Sourced in Japan
The LAS-3000 is a luminescent imager designed for detecting and analyzing chemiluminescent and fluorescent signals in various applications. It captures high-resolution images of gels, blots, and other samples using a sensitive CCD camera and a selection of excitation and emission filters.
Automatically generated - may contain errors
Lab products found in correlation
Beadbug microtube homogenizer, by Benchmark Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
2 protocols using las 3000 luminescent imager
1
Colon Protein Profiling via Cytokine Array
2
Immunoblotting Analysis of IBAV-Infected HmLu-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HmLu-1 cells were prepared in 6-well plates and infected with IBAV at an MOI of 0.01 or
3. At 12, 24, 36 and 48 hpi, cells were washed with phosphate buffered saline (PBS), lysed
with cold lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM
pyrophosphate, 1 mM β-glycerophosphate, 1 mM orthovanadate, 1% Triton X-100, and a
protease inhibitor cocktail [cOmplete; Sigma-Aldrich]), sonicated for 2 min, and then
supernatants were mixed with sample buffer and boiled for 5 min. Samples were subjected to
SDS-PAGE with 10 or 15% acrylamide gels and transferred to polyvinylidene difluoride
(PVDF) membranes (Immobilon-P; Merck-Millipore, Burlington, MA, U.S.A.). Membranes were
treated with blocking buffer (Tris-buffered saline and 0.1% Tween 20 containing 2% skim
milk). Proteins of interest were detected with specific primary and secondary antibodies
by using EzWestLumi plus (Atto, Tokyo, Japan) as the substrate. Images were taken with the
LAS 3000 luminescent imager (Fuji Film, Tokyo, Japan) and proteins were quantified using
ImageJ software version 1.52b (National Institutes of Health, Bethesda, MD, U.S.A.).
3. At 12, 24, 36 and 48 hpi, cells were washed with phosphate buffered saline (PBS), lysed
with cold lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM
pyrophosphate, 1 mM β-glycerophosphate, 1 mM orthovanadate, 1% Triton X-100, and a
protease inhibitor cocktail [cOmplete; Sigma-Aldrich]), sonicated for 2 min, and then
supernatants were mixed with sample buffer and boiled for 5 min. Samples were subjected to
SDS-PAGE with 10 or 15% acrylamide gels and transferred to polyvinylidene difluoride
(PVDF) membranes (Immobilon-P; Merck-Millipore, Burlington, MA, U.S.A.). Membranes were
treated with blocking buffer (Tris-buffered saline and 0.1% Tween 20 containing 2% skim
milk). Proteins of interest were detected with specific primary and secondary antibodies
by using EzWestLumi plus (Atto, Tokyo, Japan) as the substrate. Images were taken with the
LAS 3000 luminescent imager (Fuji Film, Tokyo, Japan) and proteins were quantified using
ImageJ software version 1.52b (National Institutes of Health, Bethesda, MD, U.S.A.).
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