Remember tailspike does not denature in SDS unless boiled, so if you want to determine the concentration of nonnative chains you will need to boil tailspike in SDS.


(1) Get purified tailspike; the extinction coefficient is 1OD 280 = 0.983 mg

dilute to a concentration of 0.25 to 0.5 mg/ml


(2) Either boil tailspike in SDS and run separatly from the native trimer on a SDS gel or boil 1/2 in Sds. Once cold add the native but if you mix together take into acount the dilution of the addition of tailspike which will not be boiled.


(3) Load at least 3 amount ie 10 ul , 20 ul, 40 ul, on the same gel as your samples are running.


(4) Calculate the amount in each standard band loaded onto the gel

NOTE: if you add sample buffer to the now concetration of tailspike remember to take that dilution into account in your calculation.


(5) Stain the gel coomassie blue and destain, quantify the gel.

Graph a standard curve of ug/ of tailspike vs volume from the densitometer


(6) Background subtraction

((background vol / background Area) * sample area) subtracted from the volume of the sample. Subtract then the pET volumes for the gel position. There is a light band about the position of the native trimer and 72 kd species.