We used western blot analysis to detect the expression of Bax, Bcl-2 and Caspase-3 proteins in lung tissue of rats from each group. The lungs were processed as already described and lung tissues were homogenized for protein extraction. After being resuspended in homogenization buffer, the tissues were centrifuged for 10 min (12,000 rpm, 4 °C). Supernatants were collected as protein suspension. SDS-PAGE (12% resolving gels at 120 V, 5% stacking gels at 75 V) was used to separate protein samples. Then protein samples were transferred to PVDF membranes (200 mA, 1 h). The membranes were blocked with 5% nonfat dry milk followed by incubation with different primary antibodies and β-actin (Santa Cruz, USA) overnight at 4 °C. After being washed with TBST, the membranes were incubated with secondary antibodies (Santa Cruz, USA) at 1:1000 dilutions for 1.5 h at room temperature. After being washed with TBST, the membranes were treated with enhanced chemiluminescence (ECL detection kit; Pierce) and exposed to the Odyssey CLx near-infrared fluorescence imaging system (LI-COR, USA). Results were quantified by AlphaEaseFC software (Alpha Innotech, USA). We performed experiments in triplicate to ensure reproducibility.
Odyssey clx near infrared fluorescence imaging system
Manufactured by LI COR
Sourced in United States
The Odyssey CLx Near-Infrared Fluorescence Imaging System is a laboratory instrument designed for high-performance fluorescence detection and imaging. It utilizes near-infrared fluorescence technology to capture and analyze fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (4 mentions)
Irdye 680cw goat anti mouse igg, by LI COR (4 mentions)
Image studio software, by LI COR (4 mentions)
Anti protein a antibody, by Merck Group (2 mentions)
Anti puromycin, by Merck Group (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Papanicolaou reagent, by Merck Group (2 mentions)
Hek293t cells, by Takara Bio (2 mentions)
Digitonin, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Bymycoprobe mycoplasma detection kit, by R&D Systems (2 mentions)
Eitheranti flag m2 affinity gel, by Merck Group (2 mentions)
Anti vsv g agarose conjugate, by Merck Group (2 mentions)
Anti vsv g antibodies, by Thermo Fisher Scientific (2 mentions)
A1978, by Merck Group (2 mentions)
Ab11174, by Abcam (2 mentions)
Ab56416, by Abcam (2 mentions)
Xt mops, by Bio-Rad (2 mentions)
20 protocols using odyssey clx near infrared fluorescence imaging system
1
Quantifying Apoptosis Proteins in Lung Tissue
2
Protein Expression Analysis in HCT-15 Cells
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The HCT-15 cells were lysed in lysis buffer containing protease inhibitor. Around 45 μg of protein from each sample was diluted to 10% and transferred to polyvinylidene difluoride (PVDF) membranes. Dried skimmed milk powder was used to block the membranes at room temperature for 1 h. The membranes were treated with primary antibodies at 4°C overnight. The membranes were incubated with secondary antibodies, and the signal was detected using the Odyssey CLx Near-Infrared Fluorescence Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Actin was used as the control.
3
Tandem Affinity Purification of Trypanosome Proteins
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For expression of N-terminal tandem affinity purification (TAP) tagged proteins, fragments of ZC3H41 (Tb927.11.1980) or Z41AP (Tb927.7.7460) open reading frames (ORFs) were cloned into p2676 [36 (link)] to yield pGR307 and pGR315, respectively. These plasmids were transfected into procyclic 449 cells and selected in the presence of 1 µg/ml puromycin. Oligonucleotides used for cloning are described in Additional file 1 . Detection of TAP-tagged proteins was carried out by immunoblot analysis using Papanicolaou reagent (Sigma) or an anti-protein A antibody (Sigma), whereas 4xTy-tagged protein levels were monitored using a BB2 monoclonal antiserum [37 (link)]. Other antisera used were anti-α-tubulin (clone B-5-1-2; Sigma), anti-p34/p37 (NRBD1/2; [38 (link)]), anti-P0 [39 (link)], anti-S9 [unpublished; a kind gift from Christine Clayton (Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg)], anti-DRBD3 [40 (link)], anti-RRP4 [6 (link)] and anti-puromycin (clone 12D10; Sigma). To prepare total cell extracts for immunoblot analysis, cells were harvested from logarithmic cultures, lysed in Laemmli buffer, boiled, and loaded directly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (1–2 × 106 cells/lane). Proteins were visualized using either chemiluminescence or an Odyssey CLx Near-Infrared Fluorescence Imaging System (LI-COR Biosciences).
4
Co-Immunoprecipitation of IR and LRP1
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HL-1 cells were cultured as above, they were treated with or without aggLDL (100 μg/mL) for 8 h and then stimulated or not with 100 nM of insulin for 30 min. Cell protein extracts were prepared using RIPA lysis buffer and incubated for 2 h at 4 °C with rabbit anti-LRP1 monoclonal antibody or rabbit non-immune IgG as IP control (2 μg/200 μg of total proteins). Then, these were incubated overnight at 4 °C with protein A-conjugated agarose beads following the manufacturer’s procedure (sc-2001; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the proteins were separated and treated for Western blot using rabbit anti-IR (1/1000) and rabbit anti-LRP1 (1/10,000) monoclonal antibodies. Secondary antibodies raised in goat anti-mouse IgG IRDye® 680CW and goat anti-rabbit IgG IRDye® 800CW (LI-COR Biosciences, Lincoln, NE, USA) diluted 1/10,000 for 1 h at room temperature. The specific bands were revealed using Odyssey CLx near-infrared fluorescence imaging system (LI-COR) and were quantified by densitometric analysis using Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA). As loading control of total protein extracts β-actin was used.
5
Western Blot Analysis of Protein Extracts
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HL-1 cells were cultured as above and cell protein extracts were prepared using RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 mM sodium ortho-vanadate, and protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). Forty micrograms of cell protein extracts were diluted in sample buffer 5X with DTT (dithiothreitol) and then heated for 5 min at 95 °C. Electrophoresis on 10% SDS-polyacrylamide gels was applied and proteins were electrotransferred to a nitrocellulose membrane (GE Healthcare Life Science, Amsterdam, The Netherlands). Nonspecific binding was blocked with 5% non-fat dry milk in a Tris-HCl saline buffer containing 0.01% Tween 20 (TBS-T) for 60 min at room temperature. The membranes were incubated overnight at 4 °C with diluted primary antibodies and secondary antibodies raised in goat anti-mouse IgG IRDye® 680CW and goat anti-rabbit IgG IRDye® 800CW (LI-COR Biosciences, Lincoln, NE, USA) diluted 1/10,000 for 1 h at room temperature. The specific bands were revealed using Odyssey CLx near-infrared fluorescence imaging system (LI-COR Biosciences, Lincoln, NE, USA).
6
Western Blot Analysis of Bacterial Proteins
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Bacterial strains were suspended in PBS, then 8 × 106 cells were mixed with SDS-PAGE sample buffer and boiled for 10 min. The samples were then run by electrophoresis on a 10% SDS gel followed by semi-dry transfer onto PVDF membrane. The membranes were blocked with 5% skim milk in tris-buffered saline/Tween (TBST; 50 mM Tris, 150 mM NaCl, 0.1% Tween 20; pH 7.6) for 3 h and incubated with 1 μg/mL of B5 mAb at 4°C overnight. Goat anti-mouse IgG (H+L) cross-absorbed secondary antibody, DyLight 680 (Invitrogen) was used at 1:5,000 dilution to facilitate detection via fluorescent signal. The final development of the signal was detected using the Odyssey CLx Near-Infrared Fluorescence Imaging System (LICOR) and analyzed using the Image Studio software (LICOR).
7
Ret and Gas1 Protein Complex Analysis
8
Plaque Assay in MDCK Cells
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All plaque assays were performed in MDCK cells using 0.6% agarose overlay. Cells were stained with either 0.1% crystal violet solution (20% methanol). When plaques needed to be visualized by immunofluorescence, cells were fixed with 10% neutral buffered formalin followed by permeabilization with PBS (0.2% Triton X-100), then incubated with mouse monoclonal α-NP (1:2,000; Iqbal laboratory). Primary antibody was detected with goat anti-mouse IgG 568-conjugated secondary antibody (1:10,000; LICOR) Plates were imaged using an Odyssey Clx near-infrared fluorescence imaging system (LICOR). ImageJ was used to measure and analyze plaques.
9
Western Blot Analysis of Autophagy Markers
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About 50 μg protein was loaded on Any kDa™ Mini-PROTEAN® TGX™ Precast protein gels and transferred to low-autofluorescence polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). For autophagy Western blots, cells were treated with a 100 nM Bafilomycin A1 (B1793; Sigma-Aldrich St. Louis, MO, USA) 4 h before collection to accumulate LC3B in the cytoplasm. Primary antibodies were γH2A.X (1:1,000; ab11174; Abcam, Cambridge, UK), cyclin-dependent kinase 2 (Cdc2) (1:1,000; 28439S; Cell Signaling Technology, Danvers, MA, USA), cyclin B1 (1:1,000; 4138S; Cell Signaling Technology, Danvers, MA, USA), p21 (1:1,000; ab188224; Abcam, Cambridge, UK), Bax (1:1,000; ab3191; Abcam, Cambridge, UK), Bcl-2 (1:1,000; ab16904; Abcam, Cambridge, UK), caspase 3 (1:1,000; ab188224; Abcam, Cambridge, UK), SQSTM1/p62 (1:1,000; ab56416; Abcam, Cambridge, UK), LC3B (1:1,000; PM036; MBL International Corp., Woburn, MA, USA), and β-actin (1:10,000; A1978; Sigma-Aldrich St. Louis, MO, USA). Immunoblots were detected with 1:10,000 goat-anti-mouse DyLight™ 680 conjugated or donkey-anti-rabbit DyLight™ 800 conjugated using an Odyssey CLx Near-Infrared Fluorescence Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
10
Western Blot Analysis of Brain Tissue
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Brain tissue was prepared for Western blotting, as described previously [27 (link)]. Briefly, tissue homogenates were prepared in RIPA lysis buffer containing protease and phosphatase inhibitors. Protein samples from each genotype and treatment group were electrophoresed through a 26 well NuPAGE 4–12% Bis-Tris Gel with MOPS SDS running buffer (Invitrogen) and transferred to polyvinylidene difluoride membranes (Immobilon-FL, Millpore). Membranes were probed using the transferrin receptor antibody (1:1,000 ThermoFisher) and α-tubulin (Sigma) as a loading control. Secondary antibodies (ferritin: IRDye® 800CW, tubulin: IRDye® 680RD) were imaged with an Odyssey® CLx near-infrared fluorescence imaging system (Li-Cor Biosciences). Immunoreactive bands were manually outlined, and densities were measured using Image Studio Lite software (Li-Cor Biosciences). The densities of immunoreactive bands were expressed as a fraction of tubulin in the same lane. Samples were run in duplicate per mouse, and the data were normalized to the WT-Veh group of that gel then averaged.
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