centrifugation the troponin c in the supernatant was dialyzed against storage buffer overnight then centrifuged if necessary to remove any particulate matter the sample was concentrated in an amicon filter to 10 ml per liter equivalent cell culture column then was loaded on pre equilibrated bio gel p60 size exclusion column the protein was eluted with storage buffer fractions were checked by 15 sds page and the fractions containing recombinant human cardiac troponin c pooled in order to remove endotoxin from the protein preparation following triton x 114 phase separation procedures are performed triton x 114 100 was added at 1 ml per 100 ml of eluate and the mixture was stirred at 4 u00b0 c for 30 min then incubated at 37 u00b0 c for 10 min the mixture was then centrifuged at 20 000 g for 5 min using 40 ml centrifuge tubes and the upper phase aspirated carefully to avoid taking the detergent phase aqueous phases were combined and the detergent step repeated twice more the aqueous phase was dialyzed against dialysis buffer overnight protein concentration was determined using the bradford assay with bsa as the standard the recombinant human cardiac troponin c was lyophilized and stored at 20 u00b0 c the following reagents were used 1 0 mg ml recombinant human cardiac troponin i 1 0 mg ml recombinant human cardiac troponin c and 1 0 mg ml recombinant human cardiac troponin t purified as described in the above examples 2 sodium dodecyl sulfate 400 mm cacl 2 400 mm mgcl2 final buffer 20 mm tris hcl 500 mm nacl 60 mm 2 mercaptoethanol ph 7 5 storage buffer 10 mm phosphate buffered saline ph 6 8 3 normal bovine serum.