|Product Overview:||The DH/PH domain of human Tiam1 protein has been produced in a bacterial expression system. It contains an MBP fusion at its amino terminus.|
|Form:||White lyophilized powder.|
|Bio-activity:||The biological activity of Tiam1 DHPH can be determined from its ability to catalyze nucleotide exchange on Rac1 using the nucleotide exchange assay of Bodipy-GDP for excess GDP or GTP. Rac1 protein is pre-loaded with Bodipy-FL-GDP by adding excess EDTA e.g. 0.7 mmol EDTA per mmol Mg2+ ions present in the reaction. This sub-stock solution is then used in a dissociation assay format which indicates competition for exchange site with unlabeled nucleotide. The reaction is monitored by fluorescence measurement at 485nm Ex / 535nm Em. Stringent quality control ensures that the exchange rate of Bodipy-GDP or mant-GDP is enhanced at least five fold in the presence of 0.8 µM Tiam1 DHPH.|
|Molecular Mass:||86 kDa|
|Purity:||Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gradient gel. MBP-Tiam1 was determined to be 90% pure.|
|Storage:||The protein is stable for six months if stored at -70 centigrade.|
|Storage Buffer:||1. MBP-Tiam1 DH/PH protein.
2. Rac1 protein.
3. Exchange buffer 2 (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 2 mM EDTA, 100 µg/ml BSA, and 0.75 µM Bodipy-FL-GDP), note - make fresh.
4. 50 mM MgCl2 in 20 mM Tris-HCl pH 7.5, 50 mM NaCl.
5. 5 mM GTP in 20 mM Tris-HCl pH 7.5, 50 mM NaCl.
|Reconstitution:||Before reconstitution, briefly centrifuge to collect the product at the bottom of the tube. The protein should be reconstituted to 5 mg/ml with ice cold nanopure water (20 µl water per 100 µg protein). When reconstituted, the protein will be in the following buffer: 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2, 5% (v/v) sucrose and 1% (v/v) dextran. In order to maintain high biological activity of the protein it is strongly recommended that the protein solution be supplemented with DTT to 1 mM final concentration, aliquoted into "experiment sized" amounts, snap frozen in liquid nitrogen and stored at -70centigrade. The protein must not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4centigrade desiccated (<10% humidity) for one year. Further Tiam1 dilutions should be made in 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2 (not supplied).|
|Official Symbol:||TIAM1 T cell lymphoma invasion and metastasis 1 [ Homo sapiens (human) ]|
|Synonyms:||TIAM1; T cell lymphoma invasion and metastasis 1; TIAM-1;|