Cells were collected at indicated times post transfection. Cells were first washed with D-PBS, then lysed in ice-cold lysis buffer (100 mM NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 0.5% Nonidet P-40, protease and phosphatase inhibitor cocktail [Roche]). For cell free virus analyses, cells were washed twice with ice-cold D-PBS, treated with 0.1% trypsin–0.02% EDTA 2 × for 5 min at 37°C, and washed twice with D-PBS prior lysis buffer (Yao et al., 1998 (link)). Cell lysates were quantified using the Bradford assay (Bio-Rad), used according to manufacturer instructions, and 20 µg of total cell lysates were denatured in Laemmli sample buffer and incubated for 5 min at 95°C prior to loading into SDS-PAGE protein separating gels. Gels were transferred onto polyvinylidene difluoride membranes (company). Membranes were blocked with 5% non-fat milk in Tris-buffered saline and 0.5% Tween 20 (TBST) prior to incubation with primary antibodies listed. Membranes were washed 3 times with TBST, and then probed with secondary HRP-conjugated secondary antibodies listed. Proteins of interest were detected using the Western Lightning Plus-ECL reagent kit (Perkin-Elmer). Signal intensity was quantified by ImageJ (NIH).
Western lightning plus ecl reagent kit
Manufactured by PerkinElmer
The Western Lightning Plus-ECL reagent kit is a chemiluminescent detection system used for western blotting applications. The kit contains reagents that facilitate the detection of protein targets on a membrane after electrophoretic separation and transfer.
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3 protocols using western lightning plus ecl reagent kit
1
Cell Lysis and Protein Extraction Protocol
2
Western Blot Analysis of PirB and PirA
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Western blot was performed as described in Kumar et al. (2020a) (link). In brief, an overnight culture of 5HP was adjusted to OD600 = 1.5, diluted (1:100) with TSB medium, and added to tubes containing taurocholate to achieve 1.5 ml aliquots with final taurocholate concentrations of 0.007, 0.03, 0.06, 0.12, 0.5, 1, and 2 mM. At 3 and 16 h, cells were harvested by centrifugation (8,000 × g for 10 min) at 4°C. Bacteria pellets (P) were resuspended with 1.5 ml PBS, and 10 μl of the P suspension and supernatant were separately run on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. Membranes were initially probed with primary antibodies for PirBvp (~51 kDa) and PirAvp (~12 kDa) in 2% bovine serum albumin (BSA) in Tris buffered saline tween-20 (TBST) for 1 h at room temperature. Then, blots were stained with secondary antibody conjugated with horseradish peroxidase (HRP). Signals were detected using a luminol-based Western Lightning Plus-ECL reagent kit (Perkin Elmer) and the intensities were detected using an ImageQuant LAS 4000 mini (GE Healthcare Life Science). ImageJ software (NIH, Bethesda, MD, United States) was used to quantify intensity of bands in immunoblots.
3
Cell Lysis and Protein Extraction Protocol
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