Western blot Cells have been harvested and lysed on ice for 45 min in RIPA lysi

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Western blot Cells have been harvested and lysed on ice for 45 min in RIPA lysi

Mensagem  Xwhk1130 em Qua Maio 18, 2016 11:17 pm

Western blot Cells have been harvested and lysed on ice for 45 min in RIPA lysis buffer. The concentration of protein was measured by Nanodrop. Equal amounts of complete protein extracts have been loaded and separated in 10% 15% SDS Webpage gel and transferred JAK 阻害剤 to PVDF membranes. The membranes have been blocked in Tris buffered saline Tween twenty with 5% milk for one hour and incubated overnight at four C with dif ferent principal antibodiesmouse monoclonal anti FLCN at a dilution of one one thousand, rabbit polyclonal anti LC3 III, rabbit polyclonal anti p62, rabbit mono clonal anti cleaved caspase 3 antibody. mouse polyclonal anti MEK, rabbit polyclonal anti phos pho MEK. rabbit polyclonal anti phospho ERK or mouse monoclonal anti Beclin 1.<br><br> The membranes had been washed in TBST and incubated with secondary antibody at room temperature for two hours. Proteins were detected with ChemiDoc detection procedure. DAPI stain and TUNEL assay Cell apoptosis was detected working with DAPI stain and TUNEL assay. Cells with indicated purchase LDE225 reagents remedy were fixed with methanolacetone for 5 min at space tem perature, then washed with phosphate buffered saline and stained with DAPI for 10 min. The cells were subsequently rinsed with PBS and observed under a fluorescent microscope. To perform the TUNEL assay, monolayer cells in 96 very well plate have been taken care of with corresponding reagents and cultured at 37 C. Cells had been subsequently fixed in 3. 7% paraformaldehyde for 7 minutes, and quantitation of apoptotic cells was measured by in situ colorimetric TUNEL assay following the producers protocol.<br><br> The outcomes had been LY2109761 臨床試験 instantly analyzed at 450 nm during the microplate reader. Autophagy assay Autophagy was detected by transmission electron micros copy, GFP LC3 and MDC assays. For transmission elec tron microscopy assay, cells were trypsinized, fixed for 24 hours with two. 5% glutaraldehyde in 0. one M sodium caco dylate, then fixed for another thirty minutes with 1. 0% osmium tetroxide. Cells were trapped in agarose, taken care of with 0. 5% uranyl acetate for 1 hour from the dark and dehy drated inside a graded series of ethanol. They have been transi tioned to propylene oxide, infiltrated in EponAraldite resin for 24 hours, embedded in molds and polymerized for 48 hours at 70 C. Blocks have been cut to determine spot into 70 nm sections.<br><br> The thin sections had been collected on mesh nickel grids and stained with aqueous uranyl acetate and lead citrate. Grids had been examined and photographed having a H 800 transmission electron microscope. For GFP LC3 assay, cells have been cultured in six very well plates and transfected with GFP LC3 with Lipofectamine 2000 following the producers protocol. At 24 hrs after transfec tion, the cells have been handled with paclitaxel or DMSO handle and cultured at 37 C for 24 hrs. The cells were subsequently examined underneath the fluores cence microscope, with 395 nm excitation wave length and 509 nm emission filter respectively. For MDC assay, cells cultured in 6 very well plate were handled with 0. 05 mM MDC and incubated at 37 Cfor twenty minutes. Soon after staining, cells have been fixed in 4% para formaldehyde for ten minutes and intracellular autophagy was detected applying a fluorescence microscope with 380 nm excitation wavelength and 525 nm emission filter.

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