Cells were reverse-transfected with siRNAs in 6-well plates as described above and 48 h later protein was isolated in RIPA buffer and quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Protein expression was measured by SDS-PAGE electrophoresis and Western immunoblotting with a c-Myc antibody (D3N8F; Cell Signaling, Danvers, MA) diluted 1:1000 in blocking buffer overnight at 4°C. Membranes were stripped before probing with a Beta-actin (β-actin) antibody (AC-74, Sigma Aldrich, St. Louis, MO) as a control for protein loading. Chemiluminescence (Clarity Western ECL substrate, Bio-Rad), was used to image bands using the Gel Logic 2200 Imaging system (Kodak, New York, USA). (See supplementary information for detailed methods).
Gel logic 2200 imaging system
Manufactured by Kodak
Sourced in United States
The Gel Logic 2200 Imaging System is a laboratory equipment designed for the capture and analysis of gel-based samples, such as DNA, RNA, and protein gels. It features a high-resolution camera and advanced imaging software to provide clear and detailed images of electrophoresis gels. The system is capable of capturing, processing, and analyzing a wide range of gel-based samples, making it a versatile tool for various scientific and research applications.
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Lab products found in correlation
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Novex see blue plus 2, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Pvdf membranes, by Jackson ImmunoResearch (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ep1556y, by Abcam (1 mentions)
Mouse monoclonal antibody against beta actin, by Merck Group (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
A2066, by Merck Group (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Pvdf membrane, by Pall Corporation (1 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions)
Molecular imaging software, by Kodak (1 mentions)
Multigene gradient thermal cycler, by Labnet (1 mentions)
14 protocols using gel logic 2200 imaging system
1
Western Blot Analysis of c-Myc Protein
2
Gel Shift Assay for Peptide/pDNA Complexes
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Gel shift assay was performed following the protocol described (Ding et al., 2019 (link); Zhang et al., 2019 (link); Geng et al., 2020 ). 1 μg of plasmid pdsRed DNA was mixed with serial N/P (nitrogen to phosphate) ratios of peptides, and the mixture was incubated for 30 min at room temperature. After adding 3 μL of loading buffer, DNA migration was measured by using 1% agarose gel electrophoresis. Imaging was performed using Kodak Gel Logic 2200 Imaging System.
To determine the stability of peptide/pDNA complex, 50% serum was added into the mixture for another 4 h. Gel retardation was visualized using a gel imaging system.
To determine the stability of peptide/pDNA complex, 50% serum was added into the mixture for another 4 h. Gel retardation was visualized using a gel imaging system.
3
Plasmid DNA Condensation by CPP-Dot1l
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The plasmid DNA condensation capability of CPP-Dot1l was examined by agarose gel retardation assay. Agarose gel separation was performed in 1× Tris-acetate-EDTA (TAE) buffer. Dot1l peptide was gently mixed with pcDNA3.1-GFP (1 μg) at indicated nitrogen to phosphate ratios (N/P) ratios in Milli-Q water or 50% serum at RT for 30 min. Afterwards, the peptide/pDNA mixture was separated by 1% agarose gel. Images were captured using the Kodak Gel Logic 2200 Imaging System.
4
Western Blotting Analysis of MAPK Signaling
5
Quantification of KCNMA1 mRNA Levels
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Cells were transfected with miR-17-5p or control mimic and AGO2 protein was immunoprecipitated as previously described [46 (link), 47 (link)]. Total RNA isolation was carried out using TRIzol reagent, followed by RT-PCR with primers specific for KCNMA1 mRNA. PCR products were run on 2 % agar gel and visualized following ethidium bromide staining. Imaging was carried out using a Gel Logic 2200 Imaging System (Kodak) under non-saturating conditions. Densitometry of band intensity was carried out using the same software.
6
Baseline SR-BI Expression in Cancer Cells
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This experiment was performed in order to assess the baseline expression of SR‐BI, the receptor for our HDL‐mimicking nanoparticles (HPPS), in different cancer cell lines. The expression of SR‐BI on the cell surface is a requirement for uptake of HPPS. KB, HT1080, Hep3B, SNU398, and Huh7 HCC cell lines were seeded on a six‐well plate at a density of 2 × 105 cells per well and grown for 24 hours. Cell lysates were prepared with radio immunoprecipitation assay buffer plus complete protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined by bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL) and immunoblotted using antibodies specific for SCARB1 (anti‐SR‐BI antibody EP1556Y, 1:1,000; Abcam, Inc.). Immunoreactive proteins were detected using goat anti‐mouse horseradish peroxidase‐conjugated secondary antibody (GenScript, Pascataway, NJ) and Clarity Western enhanced chemiluminescence (Bio‐Rad Laboratories Ltd., Ontario, Canada). Membranes were stripped and immunoblotted with a mouse monoclonal antibody against beta‐actin (1:5,000; Sigma). Imaging was carried out using a Gel Logic 2200 Imaging System (Kodak, Rochester, NY).
7
Quantifying Fatty Acids and Protein Digestion
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The amount of free fatty acids in the digesta after GI digestion was estimated as described previously (Corte-Real, Desmarchelier, Borel, Richling, Hoffmann, & Bohn, 2018) , based on the titration against sodium hydroxide (NaOH 0.1 M), using phenolphthalein as an indicator.
Sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) was carried out to study completeness of protein digestion. Samples were denatured at 95 o C for 5 min. in Lämmli buffer.
About 22 µg of protein per pocket was loaded to a 15% acrylamide and 0.4% bisacrylamide gel (gastro-intestinal digestion) and 45 g of protein per pocket was loaded to a 10% acrylamide/0.3% bisacrylamide gel (gastric digestion). Marker was Invitrogen novex see blue plus 2 (Invitrogen, Carlsbad, CA). Gel was run at 80 V (30 min.) plus 100 V (90 min.). Running buffer contained SDS (0.1%), glycine (190 mM), tris-HCl (25 mM). Fixation/staining was done with methanol/acetic acid/coomassie brilliant blue R (50%, 10%, 0.1%) (30 minutes), destaining in methanol/glacial acetic acid/water (30%, 10%, 60%, 3 h). Pictures were taken in a Kodak Gel Logic 2200 Imaging System (Kodak, Rochester, NY).
Sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) was carried out to study completeness of protein digestion. Samples were denatured at 95 o C for 5 min. in Lämmli buffer.
About 22 µg of protein per pocket was loaded to a 15% acrylamide and 0.4% bisacrylamide gel (gastro-intestinal digestion) and 45 g of protein per pocket was loaded to a 10% acrylamide/0.3% bisacrylamide gel (gastric digestion). Marker was Invitrogen novex see blue plus 2 (Invitrogen, Carlsbad, CA). Gel was run at 80 V (30 min.) plus 100 V (90 min.). Running buffer contained SDS (0.1%), glycine (190 mM), tris-HCl (25 mM). Fixation/staining was done with methanol/acetic acid/coomassie brilliant blue R (50%, 10%, 0.1%) (30 minutes), destaining in methanol/glacial acetic acid/water (30%, 10%, 60%, 3 h). Pictures were taken in a Kodak Gel Logic 2200 Imaging System (Kodak, Rochester, NY).
8
Western Blot Analysis of Monocyte Proteins
9
Bisulfite-Conversion and Methylation-Specific PCR
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MSP was performed as described previously [64 ] using primers specific for unmethylated and methylated miR-193a-3p promoter. Briefly, 1 μg DNA was bisulfite converted using the Zymo Research DNA Modification Kit (Zymo Research, Orange, CA, USA) with conversion for 18 h at 52°C, and elution in 25 μl elution buffer. MSP for unmethylated (202 bp) and methylated (198 bp) fragments was performed using 2 μl bisulfite-converted DNA as template mixed with 1x AmpliTaq Gold PCR buffer, 250 μM dNTPs, 2mM MgCl2, 400 nM forward and reverse primer and 1 unit AmpliTaq Gold DNA polymerase (all Life Technologies). PCR reactions were run on a MultiGene Gradient thermal cycler (Labnet International Inc, Woodbridge, NJ, USA) with 10 min initial denaturing at 95°C followed by 40 cycles of 1 min 95°C denaturing, 1 min 60°C annealing, 1 min 72°C elongation, followed by 10 min final elongation at 72°C. The resultant products were separated on non-denaturing polyacrylamide gels and visualized following ethidium bromide staining using a Kodak GelLogic 2200 imaging system coupled with the Kodak Molecular Imaging software (Integrated Sciences, NSW Australia).
10
Western Blotting of TLR2 and TLR4 in ESCs
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Western blotting of TLR2 and TLR4 in ESCs was performed according to the protocol published recently with some modifications (25 (link)). Blots were incubated for 2 hr with agitation in goat anti-TLR4 antibody (R&D, USA) at 1 μg/ml or 1:100 dilution of goat anti-TLR2 (Santa Cruz, USA) followed by 45 min incubation in HRP-conjugated rabbit anti-goat Ig (Sinabiotech, Iran). Bands were detected using an ECL Western blotting substrate kit and digital images were obtained with a Gel Logic 2200 imaging system (Kodak, Tokyo, Japan). Membranes were later stripped using Western Re-Probe (Calbiochem, SanDiego, CA, USA), re-incubated for 2 hr with agitation in polyclonal rabbit anti-beta actin antibody (Sigma) as loading control followed by 45 min incubation in HRP-conjugated sheep anti-rabbit Ig (Sinabiotech, Iran) and processed as above. In negative control blots, primary antibodies were substituted by equivalent dilutions of species-matched normal sera. Band densities of PCR and Western blot experiments were quantified as described elsewhere (25 (link)).
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