The cell samples from CHO-S and HEK293T cells were harvested and washed twice with PBS and lysed with RIPA buffer (Pierce, Appleton, WI). The concentration of lysate was measured using the BCA assay (Pierce, Appleton, WI). Equal amounts of lysate protein were run in a 10% Bis-Tris NUPAGE gel (Invitrogen, Carlsbad, CA) and were then transferred to a PVDF membrane using an iBlot2 machine (Invitrogen, Carlsbad, CA). Immunoblots were incubated using an anti-GFP rabbit primary antibody (Abcam, Cambridge, MA) and anti-rabbit IgG-HRP secondary antibody (Abcam, Cambridge, MA). Blots were incubated with Supersignal extended substrate (Thermo Fisher, Waltham, MA) and then imaged with an iBright CL1000 system (Invitrogen). Anti-alpha-tubulin antibody (Abcam, Cambridge, MA) was used as the loading control for both cell lines.
Ibright cl1000 system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The iBright CL1000 system is a compact and versatile imaging system designed for diverse applications in life science research. It captures high-quality images of chemiluminescent, fluorescent, and colorimetric samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti rabbit igg hrp, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Iblot2 machine, by Thermo Fisher Scientific (1 mentions)
Anti alpha tubulin antibody, by Abcam (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp secondary antibody, by Abcam (1 mentions)
Ethidium bromide, by Promega (1 mentions)
Qiaamp ucp pathogen mini kit, by Qiagen (1 mentions)
Agarose le, by Thermo Fisher Scientific (1 mentions)
Sybergold, by Thermo Fisher Scientific (1 mentions)
Kod plus neo, by Toyobo (1 mentions)
Bicinchoninic acid assay, by Merck Group (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Phospho stat6, by Cell Signaling Technology (1 mentions)
Stat6, by Cell Signaling Technology (1 mentions)
T per tissue extraction buffer, by Thermo Fisher Scientific (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Restore plus stripping buffer, by Thermo Fisher Scientific (1 mentions)
7 protocols using ibright cl1000 system
1
Protein Expression Analysis in CHO-S and HEK293T
2
Molecular Profiling of Avian E. coli
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PCR was used to differentiate non-pathogenic and avian pathogenic E. coli based on APEC-associated virulence genes. First, DNA was extracted from pure cultures of E. coli using QIAamp® UCP pathogen miniKit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions and then used to run a multiplex PCR assays targeting 8 virulence genes (namely, ompT, hlyF, iroN, tsh, vat, iss, cvi/cva, and iucD associated with APEC using previously published primers [30 (link)]. A detailed description of the PCR condition is summarized in supplementary Tables S1 and S2 . Amplified products were subjected to electrophoresis in 1.2% agarose (Agarose-LE, Ambion®, Pittsburgh, PA, USA), stained with ethidium bromide (Promega, Madison, WI, USA), and visualized using iBright CL 1000 system (Invitrogen, Waltham, MA, USA).
3
Quantitative PCR and Gel Imaging Protocol
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RT-PCR analyses were performed as described elsewhere [39 (link)]. At least three biological replicates were used for quantitative PCR, which was performed with a StepOnePlus real-time PCR detection system (Applied Biosystems; Waltham, MA, USA). Mouse Gapdh gene were used as internal control, and results were calculated using the ΔΔCT method. Semi-quantitative long PCR assays were performed in a total volume of 25 µL containing optimized PCR buffer for KOD-plus-Neo (Toyobo; Tokyo, Japan). Visualized gel image with SyberGold (Invitrogen; Carlsbad, CA, USA) was obtained by iBright CL1000 system (Invitrogen).
4
Phosphoprotein Analysis of Signaling Pathways
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Whole cell lysates were prepared and quantified using the bicinchoninic acid assay (Sigma Aldrich, St. Louis, MS, USA). Proteins from each sample (20 µg) were loaded on 10% sodium dodecyl sulfate–polyacrylamide gels, transferred to nitrocellulose membranes, and blocked in 5% bovine serum albumin for 1 h at room temperature. Membranes were incubated overnight at 4 °C with primary antibodies against phospho-JAK2 (Tyr1007/1008), phospho-STAT3 (Tyr705), phospho-mTOR (Ser2448), p-p70S6K (Thr389), phospho-Akt (Ser473), JAK2, Akt (pan), mTOR, p70S6K, STAT3, and GAPDH, diluted in 1x TBST solution containing 5% BSA with 1:1000 ratio, treated with mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., Dallas, Texas, USA), and analyzed using an iBright CL1000 system (Thermo Fisher Scientific, Waltham, MA, USA) after enhancement with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA). All primary antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). The band intensity of Western blot was measured and quantified by Image J program.
5
Imaging and Visualization of Biomolecules
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The CBB-stained gels were imaged under white light using an iBright CL1000 system (Thermo Fisher Scientific) or a regular cellphone camera. The antibody-based immunoblotting membranes were imaged with the iBright CL1000 system under the chemiluminescence mode. Ni2+-trisNTAAlexa405–stained gels and membranes were exposed to the UV light generated by the UV transilluminator of the FluorChem Q Image analysis system (Alpha Innotech) and imaged with the camera of the same system or by a regular cellphone camera. The Atto550, Cy3B, Atto647N, and Alexa Fluor 647 conjugate-stained gels were imaged using an Amersham Biosciences Typhoon biomolecular laser scanner (GE Healthcare).
6
Western Blot Analysis of Cellular Signaling
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Tissues were ground and lysed with T-PER tissue extraction buffer (ThermoFisher Scientific, IL, USA) and cells were lysed with RIPA buffer (ThermoFisher Scientific, IL, USA) to harvest proteins. Appropriate protein amounts were loaded into sodium dodecyl sulfate-polyacrylamide gels and blotted onto BioTraces nitrocellulose membrane (Pall Life Sciences, FL, USA). The membranes were blocked with a 5% BSA solution and treated with primary antibodies for anti-phospho-p44/42 MAPK (ERK1/2), phospho-p38 MAPK, phospho-SAPK/JNK, phospho-STAT3, phospho-STAT6, phospho-JAK1, p44/42 MAPK (ERK1/2), p38 MAPK, JNK, STAT3, STAT6, JAK1, and GAPDH (Cell Signaling Technology, MA, USA, 1:1000). The mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., Dallas, TX, USA, Cat no. sc-2357, 1:5000) were used Images were taken using the iBright CL1000 system (ThermoFisher Scientific, IL, USA).
7
Vitamin D Signaling in Breast Cancer
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SUM159PT (700,000 cells), Hs578T (300,000 cells) and MDA-MB-453 (800,000 cells) cells were plated in 100mm dishes in standard media and treated the next day for 48 h with 1,25(OH) 2 D 3 (100 nM), K2 (10 μM) or both. Whole cell lysates (50 μg/lane) were separated by SDS-PAGE, wet-transferred to PVDF membranes, and blocked for 1h in 5% skim milk/PBS. Blots were incubated overnight with primary antibodies against VDR (D6, Santa Cruz Biotechnology, Dallas, TX), Gla (Biomedica Diagnostics, Stamford, CT), Cyclin D1 (Cell Signaling Technology, Danvers, MA) or c-Myc (Cell Signaling) and then incubated for 1h with anti-rabbit or anti-mouse ECL horseradish peroxidase-linked secondary antibody (GE Healthcare, Chicago, IL). SuperSignal West Dura Extended Duration Substrate (ThermoFisher, Waltham, MA) was used for imaging of blots on an iBright CL1000 system (ThermoFisher). After imaging, blots were stripped with Restore PLUS stripping buffer (ThermoFisher) and reprobed with GAPDH primary antibody (Bio-Rad Laboratories, Hercules, CA), followed by incubation with an anti-mouse ECL horseradish peroxidase-linked secondary antibody.
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