MTMR3 was first reported as a dual-specific phosphatase, having phosphatase activity toward phosphorylated serine, threonine, and tyrosine residues (3). MTMR3 (Myotubularin-related protein 3), also known as FYVE-DSP1, contains an amino terminal pleckstrin homology (PH) domain and a carboxyl terminal FYVE domain. Myotubularin-related proteins are a family of phosphatases with emerging roles in cellular signaling and membrane trafficking (1,2). Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Prepare 1X SignalFire™ ECL Reagent ( #6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g.Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.Detection of Proteins Directions for Use: Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody ( #7074 at 1:2000) and anti-biotin, HRP-linked Antibody ( #7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.ĭ.Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4☌.Wash three times for 5 min each with 15 ml of TBST.Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.(Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.NOTE: Volumes are for 10 cm x 10 cm (100 cm 2) of membrane for different sized membranes, adjust volumes accordingly. Membrane Blocking and Antibody Incubations Electrotransfer to nitrocellulose membrane ( #12369).Ĭ.NOTE: Loading of prestained molecular weight markers ( #59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder ( #7727, 10 µl/lane) to determine molecular weights are recommended. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm). Heat a 20 µl sample to 95–100☌ for 5 min cool on ice.Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate).Aspirate media from cultures wash cells with 1X PBS aspirate.Treat cells by adding fresh media containing regulator for desired time.Protein Blotting A general protocol for sample preparation. Detection Reagent: SignalFire™ ECL Reagent ( #6883).ī.Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody ( #7074).Pore size 0.2 µm is generally recommended. Blotting Membrane and Paper: ( #12369) This protocol has been optimized for nitrocellulose membranes.Blue Prestained Protein Marker, Broad Range (11-250 kDa): ( #59329).Biotinylated Protein Ladder Detection Pack: ( #7727).Primary Antibody Dilution Buffer: 1X TBST with 5% BSA for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.10X Tris Buffered Saline with Tween ® 20 (TBST): ( #9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH 2O, mix.10X Tris-Glycine Transfer Buffer: ( #12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH 2O, mix.10X Tris-Glycine SDS Running Buffer: ( #4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH 2O, mix.1X SDS Sample Buffer: Blue Loading Pack ( #7722) or Red Loading Pack ( #7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
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