http://cms.biomslab.org Krutchinsky lab Wed, 18 Jun 2008 18:18:39 +0000 http://backend.userland.com/rss092 en This protocol describes the I-DIRT technique that helps to distinguish contaminants from the real interactors in immunopurified complexes. Here is the original paper describing the details of the technique. PROTOCOL: 1. Prepare media Media for Isotopically heavy Yeast- Reagents for 1L mix Yeast nitrogen base without amino acids (Sigma Y0626)- 6.7 g Synthetic drop-out media ... http://cms.biomslab.org/general/i-dirt-protocol/i-dirt-a-general-method-for-distinguishing-between-specific-and-nonspecific-protein-interactions/ 1. Put ~1-2g of pellets of frozen cells in the 50 ml stain less jars previously immersed in the liquid nitrogen until it stops boiling. 2. Close the jars. Do not screw the lid tightly. 3. Insert the jars into a Retsch Mixer Mill MM301, and beat for 5 times, 3’ at ... http://cms.biomslab.org/general/cryogenic-disruption-of-yeast-cells/cryogenic-disruption-of-yeast-cells/ For one reaction use 1. 500 ug of Antibody (Antiflag-M2 Monoclonal Antibody, Sigma F-3165, 5 mg of 4mg/ml solution). Antibody should be in phosphate buffer only! Glycerol and preservatives will interfere with coupling to epoxy groups activated on the beads. 2. 50 mg of Dynal beads (M270 Epoxy, 300 mg/vail) NOTE> 50 mg ... http://cms.biomslab.org/general/coupling-ab-to-beads/protocol-for-binding-flag-m2-antibody-to-the-dynal-beads/ 1. Culture 20 flasks (Corning, 430641; 75 cm2 culture area/flask) of mammalian cells until reach over 95% confluent. 2. Remove the culture medium, and rinse the cells twice with cold 1X PBS and aspirate. 3. For each flask, add 1 ml IP Buffer (20 mM Hepes (4.79 g/L), 2 mM MgCl2 (0.19 ... http://cms.biomslab.org/general/ip-from-mammalian-cells/a-protocol-of-ip-from-mammalian-cell-cultures/ . Eisosomes We applied out methods and tools to investigate the composition and the dynamics of Eisosomes. We tagged two proteins Pil1 and Lsp1 with the our modular tags and, purified and identified several proteins. Using reciprocal tagging we confirmed that some of the identified proteins co-localize with ... http://cms.biomslab.org/projects/projects/ Use prOTOF mass spectrometer to measure the tryptic map spectra. version 20.12.06 1. The prOTOF mass spectrometer should indicate that it is ready for the analysis by a green light, "TOF Vacuum", on the front panel. 2. Wake up the prOTOF computer by pressing the computer switch button quickly. Login and type a ... http://cms.biomslab.org/general/using-modular-ms/using-modular-ms-tool/ We use double affinity tags, 3xFLAG-6xH or 3xMyc-8xH, for purification of proteins and protein complexes (see our IP protocol). The use of 3xFLAG or 3xMyc tags are usually sufficient to obtain highly enriched protein complexes. However, an additional tandem purification step can be very useful to remove an excess ... http://cms.biomslab.org/general/on-beads-digestion-protocol/on-beads-digestion-of-protein-complexes/ Version 25.5.07 All operations, at least before stopping digestion, are preferably done in a clean dust free environment (air clean workstation, clean room etc.). 1. Cut the gel band of interest. Do not cut a sharp thin pieces. The width of the gel piece should be ~1 mm or more. Note: There is ... http://cms.biomslab.org/general/in-gel-digestion-protocol/in-gel-digestion-protocol/ 1. Grow several liters of cells overnight to the density 2-5x10e7 cells/ml 2. Spin down cells in 1 liter bottles at 5000rpm 15-20’ 4ºC 3. Remove supernatant and collect cells with up to 50ml of ice cold water into 50ml conical tube 4. Spin down cells for 1’ at 4000rcf and remove the ... http://cms.biomslab.org/general/ip-protocol/a-protocol-for-purification-of-proteins-tagged-with-with-a-3xflag-6xh-double-affinity-tag/ We develop and build new mass spectrometric tools to advance our research. One recent development is a A Novel High‐Capacity Ion Trap‐Quadrupole Tandem Mass Spectrometer constructed in collaboration between our laboratory and Brian Chait's laboratory at the Rockefeller University. This mass spectrometer was built as a prove of the principle ... http://cms.biomslab.org/new-mass-spectrometers/new-ms-tools/