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reconstituting enyzmes...

enzyme reconstitution

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#1 pchu

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Posted 03 June 2013 - 01:21 PM

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!

seikagaku sigma

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

Edited by pchu, 03 June 2013 - 01:22 PM.

#2 almost a doctor

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Posted 04 June 2013 - 01:19 AM

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

#3 pchu

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Posted 04 June 2013 - 02:29 PM

Thank you for confirming, i really appreciate it!! now i know how to deal with these problems