simple question but i'm a little confused.
I'm purifying an enzyme via IEX, i need to calculate purification yield and protein recovery.
Target protein:
Load (In): 10ml x 0.18mg/ml = 1.8mg
Elution (Out): 8ml x 0.2mg/ml = 1.6mg
so 1.6/1.8 X 100 = 89% is this my recovery or yield?
i also measured HCP:
0.574 mg in LOAD
0.160 mg in FT (28%)
0.308 mg in Eluate (54%)
0.096 mg in strip (17%)
So 54% of HCP ends up in eluate together with my enzyme,
does this mean that my yield is 46% then??
Thank you!
damian
The first you calculated is protein recovery. In order to determine the enzyme yield you have to calculate total enzyme activity before loading on IEX (call this A), then calculate the total activity after elution (call this B). Then BX100/A :enzyme yield. For Purification fold first you have to find specific activity for A sample as dividing Total activity units/Total protein mgs:specific activity as Units/mg protein, then do the same for B sample. Finally divide Specific activities B/A: Purification fold. You can repeat all these after each purification step you follow.
The first you calculated is protein recovery. In order to determine the enzyme yield you have to calculate total enzyme activity before loading on IEX (call this A), then calculate the total activity after elution (call this B). Then BX100/A :enzyme yield. For Purification fold first you have to find specific activity for A sample as dividing Total activity units/Total protein mgs:specific activity as Units/mg protein, then do the same for B sample. Finally divide Specific activities B/A: Purification fold. You can repeat all these after each purification step you follow.
thank you Mr Guven,
could i calculate yield with concentration and not activity? i don't have an activity assay ready yet. how is it calculated for antibodies?
No. HPLC is mass related (ng or ug on column). In activity you can have 1 molecule performing 100's even 1000's reactions before it is exhausted. This depends on the microenvironment. of the molecule.
Bruce is absolutely correct. When you purify and isolate an enzyme, the first thing you need is an assay for that enzyme. There are two measurements that you will need to make at each stage of purification: activity and protein content. Your degree of purification will be measured by both, in terms of specific activity (U/mg), and yields will be measured in terms of total units of enzyme recovered.
HI
you need to do purification table , contain total activity of enzyme and protien concentration to calculate specific activity as Units/mg protein then calculate Purification fold and enzyme yield.
good luck
feel free to contact to me
@Greg Cote
WOW... I also have the exact same table from PL. Was a great company for cofactors, Coenzyme A derivates and nucleotides in the day, and I still have their catalogue that lists all the extinction coefficients for these things...
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