Saturday, October 18, 2003 Description This method uses carboxyfluorescein succinimidyl ester (CFSE) rather than fluorescein isothiocyanate, resulting in more reliable labeling. Succinimidyl esters are excellent reagents for amine modifica...
Thursday, October 16, 2003 Description TRANSFECTION OF MAMMALIAN CELLS USING LIPOFECTAMINE REAGENT Procedure 1. In a six-well or 35 mm tissue culture plate, seed ~2x 105 cells per well in 2 ml Basal Medium containing 10% FBS. 2. Incubate t...
Wednesday, October 01, 2003 Description Alkaline phosphatases (APs) are highly ubiquitous enzymes, present in all species from bacteria to man Procedure 1.Grow bacteria overnight in Pi sufficient and Pi deficient media. 2.Read OD600 of ove...
Friday, June 11, 2004 Description Medium throughput assay for detection of the phosphorylation of Hsp27 by upstream kinases (notably MapKap Kinase 2) Procedure Phospho-HSP27 Cellular Assay Cell Type: THP-1 cells from ATCC (TIB-202) that ha...
Sunday, December 28, 2003 Description This review discusses the EnzChek phosphatase assay kit comercially available from Molecular Probes Inc. that we have found to be very valuable to assay probable phosphatase activity of enzymes. The as...
Description Growth, Purification, and Refolding of TGF-b3 (for 3L) Procedure 1) Transform BL21(DE3)cells with pET32 plasmid containing the TGF-b3 sequence a) Place 10ml competent cells in a microfuge tube and add ~0.5mg plasmid DNAon ice....
Description Procedure for Ion (DEAE) Exchange Chromatography for ecT bR2 Purification Procedure For cells from a 3.0 liter culture: 1. Prepare 400ml of 50mM Imidazole/0.6MNaCl/pH 7.0 2. Next, set up one 45 ml bed volumecolumn of DEAE-Sepha...
Friday, November 21, 2003 Description The preparation of a biomolecule for NMR studies usually requires exchanging the sample in an aqueous solution appropriate for conducting the proposed NMR studies. The two main requirements that must b...
Friday, November 21, 2003 Description The following is a protocol for growing 1.0 L of E. coli in deuterium oxide (D2O)-M9 salts medium. Due to the slow growth of cells in D2O minimal medium, the D2O culture is inoculated with cells at 0.3...
Friday, November 21, 2003 Description Analysis of soluble/insoluble partitioning of E. coli overexpressed proteins Procedure Pellet 1.0 mL of induced cell culture in a microfuge tube. Remove supernant. Resuspend the cells in 0.5 mL of MQ H...