Samples of the quadriceps (QUA) and gastrocnemius (GAS) muscles were lysed in lysis buffer (iNtRON Biotechnology, Seoul, Korea) containing protease and phosphatase inhibitors. The total protein contents of the lysates were quantified using a BCA protein assay (Pierce, Rockford, IL, USA). Lysates containing equal amounts of protein were separated by SDS-PAGE and the separated proteins were transferred to membranes. The membranes were blocked with 5% skimmed milk and washed with Tris-buffered saline containing 0.05% Tween-20. The membranes were incubated overnight at 4 °C with the primary antibodies; PI3K, p-Akt, Akt, p-mTOR, mTOR, p-p70S6K, p70S6K, p-4EBP1, 4EBP1, myoD, myogenin, p-FoxO3a, FoxO3a, Fbx32, and MuRF1, and then with a secondary antibody (peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat antibodies; Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. The target protein bands were detected using a EZ-Western Lumi Femto kit (DoGenBio, Seoul, Korea) and imaged using a LAS-4000 instrument (GE Healthcare Life Sciences, Marlborough, MA, USA). The relative band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA).
Las 4000 instrument
Manufactured by GE Healthcare
Sourced in Sweden, United States
The LAS-4000 is a high-performance imaging system designed for life science research applications. It is capable of capturing and analyzing fluorescent, chemiluminescent, and colorimetric signals from a variety of sample types, including gels, membranes, and microplates. The instrument utilizes a sensitive CCD camera and advanced imaging optics to provide high-quality image data.
Automatically generated - may contain errors
Lab products found in correlation
Hybond c extra, by GE Healthcare (2 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (2 mentions)
Ez western lumi femto kit, by DoGenBio (1 mentions)
Lysis buffer, by iNtRON Biotechnology (1 mentions)
Horseradish peroxidase coupled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Sc 9996, by Santa Cruz Biotechnology (1 mentions)
Effectene, by Qiagen (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Na931, by GE Healthcare (1 mentions)
Ma5 11869, by Thermo Fisher Scientific (1 mentions)
Phosstop, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Blotto, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by GenDEPOT (1 mentions)
Stepone qrt pcr system, by Thermo Fisher Scientific (1 mentions)
V 550 spectrophotometer, by Jasco (1 mentions)
Bas 5000 bio imaging analyzer, by GE Healthcare (1 mentions)
7 protocols using las 4000 instrument
1
Muscle Protein Expression Analysis
2
Protein Expression Analysis of Melanoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
B16F10 cells were seeded on a 100-mm culture dish and cultured for 24 hours. These were treated with a-MSH (100 nM), TAM (5 μg/mL, 25 μg/mL, and 50 μg/mL), and kojic acid (50 ppm) and cultured for 24–48 hours. Then the culture medium was removed. After washing twice with phosphate buffered saline, RIPA buffer (100 mL) added to the complete mini 1 tab was dissolved in 100 μL. Next, it was centrifuged for 20 minutes at 4 °C, 12,000 rpm. The centrifuged supernatant was quantified by the Bradford assay, and 30 μL protein was separated by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using 10% gel. The semidry transfer cell instrument (Hoefer, Inc., San Francisco, CA, USA) was used to move the separated protein on the polyvinyl difluoride membrane. Then, it was cultured in the blocking buffer (5% skim milk in Tris-buffered saline with Tween) for 1 hour at room temperature. The primary antibody [protein kinase A (PKA), ß-actin] was diluted and kept overnight at 4 °C. It was washed using Tris-buffered saline with Tween for three times in at 10-minute intervals. The secondary antibody (anti rabbit, anti goat) was diluted by 1:1000, and cultured for 2 hours at room temperature. Then, it was washed for three times. The LAS 4000 instrument (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was used to measure the band for quantitative analysis.
3
Transient Transfection and Western Blot Analysis
4
Western Blotting for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as previously described53 (link). Tissues and cells were homogenized in ice-cold RIPA buffer containing protease inhibitor (cOmplete Protease Inhibitor Cocktail, Merck) and phosphatase inhibitor (PhosSTOP, Sigma). Supernatants were collected after centrifugation and mixed with an equal amount of loading buffer (125 mM Tris–HCl pH 6.8, 30% glycerol, 10% SDS, and 0.6 M DTT). After heat denaturation, the protein samples were subjected to SDS-PAGE using 10% acrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against phospho-p44/42 MAPK (ERK1/2) (Thy202/Thy204) (9101S; Cell Signaling Technology, Danvers, MA, USA), p44/42 MAPK (ERK1/2) (9102S, Cell Signaling), and actin (MA5-11869; Thermo Fisher Scientific), and then with secondary antibodies against rabbit IgG (7074S; Cell Signaling Technology) and mouse IgG (NA931; GE Healthcare, Little Chalfont, UK). Signals were detected with the Pierce ECL Prime system (GE Healthcare) in a LAS4000 instrument (GE Healthcare). Band densities were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Analysis of Drosophila Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heads from 1 day old flies were homogenized in 2× SDS-PAGE sample buffer followed by boiling at 95°C for 5 min. Samples were separated using SDS-PAGE and electro blotted onto nitrocellulose membrane (Hybond-C Extra; GE Healthcare) using semidry transfer assembly (Bio-Rad). Following blocking with 5% Blotto (Santa Cruz Biotechnology, CA), blots were incubated overnight at 4°C in appropriate dilutions of primary antibodies [anti-α-tubulin (1:5000 dilution; E7 DSHB), anti-Gαq (1:1000 dilution), anti-TRP (1:5000 dilution), anti-Rh1 (1:200,4C5 DSHB), anti-INAD (1:2000) and anti-NORPA (1:1000)]. Protein immunoreacted with the primary antibody was visualized after incubation in 1:10,000 dilution of appropriate secondary antibody coupled to horseradish peroxidase (Jackson Immuno Research Laboratories) for 2 h at room temperature. Blots were developed with ECL (GE Healthcare) and imaged using a LAS 4000 instrument (GE Healthcare).
6
Immunoblotting and immunoprecipitation of SAMHD1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using a lysis buffer from Cell Signaling Technology (#9803) with a protease inhibitor cocktail (Gendepot, Katy, TX, USA), and 30 μg of the lysates were used for immunoblotting. For immunoprecipitation, 900 μg protein was incubated overnight at 4 °C with the corresponding primary antibodies conjugated to G beads (MilliporeSigma) or FLAG-tagged magnetic beads (Sigma‒Aldrich). Beads were washed with a washing buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, 0.2 mM EDTA, and 0.1% Triton X-100. After SDS‒PAGE, membranes were immunoblotted using the corresponding primary antibodies and HRP-conjugated secondary antibodies. Visualization was performed using Lumi-Pico solution (Dogen, Seoul, Korea) and a LAS-4000 instrument (GE Healthcare, Chicago, IL, USA). SAMHD1 levels were quantified as integrated band intensities using ImageJ (National Institutes of Health, Bethesda, MD, USA) and normalized to the levels of the corresponding housekeeping proteins.
7
Quantitative Analysis of DHFR and sfGFP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synthesized DHFR and sfGFP containing radioactive [35S]Met were analyzed by 15% SDS-PAGE, and the gel image was visualized using a BAS-5000 bio-imaging analyzer (GE Healthcare, USA). Time-course analysis of sfGFP expression was performed by measuring sfGFP fluorescence every 3 min using a StepOne qRT-PCR system (#4376373, Applied Biosystems, USA). Fluorescence images of synthesized sfGFP in reaction mixtures were obtained using an LAS-4000 instrument (GE Healthcare, USA). The activity of synthesized DHFR was measured as described previously44 (link). Reaction mixtures (2 mL) contained 50 mM MES-KOH pH 7.0, 25 mM TRIS-HCl pH 7.0, 25 mM ethanolamine, 100 mM NaCl, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 100 μM dihydrofolic acid, and a 10 μL aliquot of the PURE reaction mixture, and they were incubated at 37°C for 15 min. Next, 20 mM NADPH was added to a final concentration of 200 μM, and the decrease in absorbance at 340 nm was measured over a period of 10 min with a V-550 spectrophotometer (Jasco, Japan). One unit of DHFR was defined as the amount of enzyme required to process 1 μmol of dihydrofolic acid in 1 min at 37 °C.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!