I am using IPTG induction to express my protein of interest. At the end of induction and cell lysis, I'll be re-suspending my pellet in 40ml buffer. My protein has a N-terminus Strep-II tag and hence I want to use strep-tactin super flow resin from IBA life sciences. However, I am really confused what volume of resin I should be using for the purification. They say the binding capacity of 1ml of resin is approximately 50nmol of 70kDa protein which is around 3.5mgs.
Hi, No one is going to put exact number for you.
The best way is after resuspending you will centrifuge and collect the supernatant after that just check absorbance or estimate the amount of protein that you might have ( Yes, it is crude extract and have many proteins, but if your protein is expressed as a major content you can use that number) and then use beads accordingly- like less than that number.
It is better to avoid using more than necessary resin. In general- 500 microliter to 2 ml is reasonable.
Dear Ramyani
it really depends from the e.coli biomass the you resuspend and from the level of the expression of the protein in this pellet.
for example a strong over expression. l from 1g of Wet biomass can provide also 5 mg of pure protein while a poor expression related to a weak band detectable on sds-page can give 0.2-0 3 mgs.
also if step tag is very specific as Mahesh told as General rule It is better to avoid using more than necessary resin not only for the cost but because if you are performing the purification in condiction close to the resin saturation yiu will increase the purity level of your final sample because the protein compete with aspecific binders for the site availabl on the resin. Mantain the flow through and run all the fraction in the sta page, so you can reload it in the coloumn and recover the unbounded protein in the case that you understimate ora amount and you have good amount in the inglese fraction
good luck
Manuele
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