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Recombinant Human TIAM1 Protein

Cat.No.: TIAM1-25H
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.
Source: E. coli
Species: Human
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).
Gene Name: TIAM1
Official Symbol: TIAM1 T cell lymphoma invasion and metastasis 1 [ Homo sapiens (human) ]
Synonyms: TIAM1; T cell lymphoma invasion and metastasis 1; TIAM-1;
Gene ID: 7074
mRNA Refseq: NM_003253
Protein Refseq: NP_003244
MIM: 600687
UniProt ID: Q13009

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