Lymphocytes form DLNs in EAE mice were stained with anti-CD4 antibodies and sorted on a FACS instrument (BD Biosciences, USA) by gating on the CD4+ population. For western blot analysis experiment, the sorted CD4+ T cells were lysed in lysis buffer (300 mM NaCl, 50 mM Tris pH 8.0, 0.4% NP-40, 10 mM MgCl2, and 2.5 mM CaCl2) supplemented with protease inhibitors (Complete mini EDTA-free; Roche Diagnostics, Mannheim, Germany). After centrifugation, the supernatant was measured using the BCA protein assay reagent (Pierce, Rockford, IL). Then, 1 μg of total cell extracts protein was loaded onto 12.5% SDS-PAGE, transferred to nitrocellulose membrane (Amersham Pharmacia Biotech, Little Chalfont, UK), blocked by incubation with 5% non-fat milk in TBS-T buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl and 0.1% Tween-20) for 1 h, and blotted against the different proteins using specific antibodies: anti-caspase-3, anti-Bax, anti-Bcl-2, anti-β-Actin and anti-cytochrome c. After washings with TBS, the protein bands were visualized using DyLight 800/DyLight 680-conjugated secondary antibodies, and the infrared fluorescence image was obtained using an Odyssey infrared imaging system (LI-COR Biosciences, USA).
Dylight 680
Manufactured by LI COR
Sourced in United States
DyLight 680 is a fluorescent dye that can be used for labeling biomolecules, such as proteins, antibodies, or nucleic acids. It has an excitation maximum at 682 nm and an emission maximum at 715 nm, making it suitable for applications that require detection in the near-infrared region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (4 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Facs instrument, by BD (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mini protean precast gel, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Dylight 800, by LI COR (1 mentions)
Native laminin, by Merck Group (1 mentions)
Pathscan intracellular signaling array kit, by Cell Signaling Technology (1 mentions)
Array analysis software, by LI COR (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
5 protocols using dylight 680
1
Immunoblot Analysis of Apoptosis Markers in CD4+ T Cells
2
Western Blot Analysis of mTOR Pathway
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LM fibroblasts cells (parental LM or ShR4-1) were lysed for 2 hr at 4°C in NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl) with 1% protease inhibitor cocktail (Sigma, P8340) and 1% phosphatase inhibitor (Sigma, P5726). 18 μg of proteins were separated using 4–20% Mini-PROTEAN precast gel (BioRad). Subsequently, these proteins were transferred onto PVDF membranes (Millipore). The membrane was blocked by 5% skim milk dissolved with a wash buffer (Tris/HCl pH 7.5, 0.05% Tween-20) for 1 hr at room temperature. The membrane was incubated with the appropriate primary antibody (listed in the corresponding table) followed by the compatible Fluorescein-conjugated secondary antibody (listed in the corresponding table). Fluorescence was detected by the Odyssey Imaging System (LI-COR Biosciences).
| Target | Merchandiser | Remarks |
|---|---|---|
| β-tubulin | CST | #86298, D3U1W |
| Mouse IgG | CST | #5470, DyLight 680-conjugated |
| Rabbit IgG | CST | #5151, DyLight 800-conjugated |
| mTOR | CST | #4517, L27D4 |
| mTOR (phosho-Ser2448) | CST | #5536 |
| S6 | CST | #2317, 54D2 |
| S6 (phospho-Ser240/244) | CST | #5364, D68F8 |
| 4-EBP1 | CST | #9644, 53H11 |
3
Laminin Overlay Assay for Protein Analysis
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The laminin overlay assay was performed with 20 μL of WGA purified samples separated on 4% to 15% SDS-polyacrylamide gels by electrophoresis and then transferred to PVDF membranes. Transfers were blocked with PBS and 5% nonfat dry milk for 1 h at room temperature, and then briefly rinsed with TBS. Membranes were incubated for 2 h at room temperature in TBS containing 1 mM CaCl2, 1 mM MgCl2 (TBSS), 3% BSA, and 1 mg/mL native laminin (L2020 Sigma). After two 10-min washes in TBSS, the membranes were incubated overnight at 4°C with TBSS 3% BSA and anti-Laminin (Table S3 ). Subsequently, membranes were washed with TBSS twice for 10 min, incubated with anti-rabbit DyLight 680 for 45 min at room temperature, washed twice with TBSS for 10 min, and then visualized using Licor's Odyssey Infrared Imaging System. As a negative control, we used TBSS without 1 mM CaCl2 during incubation and washes.
4
Intracellular Signaling Array Analysis
5
Quantitative Western Blot Analysis
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