reconstituting enyzmes... - (Jun/03/2013 )
hi so im trying to make equivalent 2 batches of enzymes from different manufacturers ( seikagaku vs. sigma ) and im totally confusing myself because the units that came in each vial is different and their specific activities are different too!
ug/vial (protein) 31.9 111.6
units/mg protein (specific activity) 183 113
units / vial 5.84 12.61
per the instructions in the seikagaku batch, i dissolved the solid in 200 ul of 0.1% BSA and made twenty 10 ul aliquots . now im not sure how to reconstitute the sigma batch while maintaing a similar amount of activity in each aliquot!
i ended up calculating the amount of protein / ul and from that determined the amount of ug protein / aliquot, then calculated the units / ug protein to figure out how many units/aliquot. does that sound right? i got ~430 ul 0.1% BSA to dissolve the sigma batch in
my advisor simply saw that there were roughly double the units in the sigma batch but only about 60% of the specific activity and did this calculation
(200ul 0.1% BSA * 2) (0.6) = 240 ul 0.1% BSA
any advise would be appreciated!
Hello, I'm sorry but your advisor is wrong. Yes, the sigma batch has 60% of the specific activity, but there's 3x more protein (111.6ug compared to 31.9ug), which is why there is roughly double the total amount of units.
I think the simplest way to solve your problem (and get the same answer you already got), is thin about TOTAL units. So,
Seikagaku = 5.84 units you've diluted this in 200ul , so now you have 5.84units/200ul = 0.0292units/ul it doesn't matter how many aliquots of which volume each, your sample is now at that concentration.
Sigma = 12.61 units to know the volume you need to dilute this to a final concentration of 0.0292units/ul you just need to divide the total units by the final concentration you want = 12.61 units/0.0292unit/ul = 431ul
Hope this helps
Thank you for confirming, i really appreciate it!! now i know how to deal with these problems