Hi,
I am about to carry out a few enzyme assays. After protein purification and dialysis to remove the salts, what is the acceptable maximum time to keep the protein at 4 degree before performing enzyme test? Or I wonder what is the common option in your lab, test before it loses activity at 4 degree or store the protein at -80 degree for long term use? I prefer to test enzyme activity immediately after preparation, but not quite sure how long the enzyme activity may sustain at 4 degree! Thanks:)))
Yong
@ Yong Jia,
The dialysed enzyme is stable for several hours at appropriate pH at 0-4C. If it looses activity, you could try DTT (dithiothretol) and EDTA to stabilize it. For long-term storage we use -80C
@Tushar
if by several hours you mean possibly several hundreds of hours, sure
@Yong
that depends both on your enzyme and on the buffer you keep it in (plus on how pure it is)
1) your enzyme may be stable or may denature very quickly. I've experienced both cases. When I was purifying my isomerase, I could have left it in the fridge for two weeks and when I would come back, there would be still at least 90%, no matter in what buffer it was. However, I started with purification of another enzyme and I was very surprised when I lost all the activity in less than one week.
2) your enzyme may be sensitive to additives, it may require certain ionic strength (so removing ALL salt is not very wise), glycerol may help to stabilize your protein or it may require reduced conditions (mercaptoethanol, DTT), especially if it's redox enzyme. Further the buffer type and strength may influence the stability
3) and of course, if it's only partly purified, there may be still some proteases which would degrade your enzyme and thus decrease the perceived stability
As mention above, its depends on your enzyme. Characteristic of the enzyme and the buffer+additives that you use.
For me, im using EDTA to chelate heavy metal contaminant that may inhibit the enzyme activity which also inhibit phenol oxidase. Also use BME as reducing agent.
By using those, I can retain my enzyme activity longer in 4 degree (3-4 days) but fresh enzyme was always the best.
Talking about retaining the enzyme activity, for storage in -80, I divide my enzyme into several small tube to reduce thawing effect. Each time I want to use it, I just have to take out an amount of enzyme that I need only. Hope that answer helps you.
Thanks all for the promt answers! Very informative:)
So it all depends, and you need to experiment to find out right? My protein is an oxido-reductase and needs Zn ion to perform its activity. Better avoid EDTA. Just one more question, when you add glycerol for long term storage, could it affect the subsequent enzyme kinetic test? Thanks
Yes, in your case it's better to add some free zinc and reducing agent. The glycerol may help (especially when the enzyme is purified and thus diluted).
It could affect the enzyme measurement, you have to try. But the enzyme should constitute only small fraction in the reaction mixture anyway and since the glycerol will be 10-20%, it should be less than 1% in the reaction.
@ Yong Jia,
With any enzyme the "acceptable time" must be determined empirically, by determining specific activity at different times post-purification/storage. This will tell you the time-dependent loss in enzyme activity.
Storage at 4oC is ok for some very robust proteins e.g. streptavidin. More commonly, proteins are stored at -20/-80oC. If your protein in sensitive to freeze-thaw cycles, I would recommend making small aliquots and storing at -80oC and then removing an aliqout and storing at -20oC for the duration of your experiments. Your storage buffer will need high glycerol content >40% to remain liquid at -20oC to avoid freeze-thaw.
When freezing proteins for storage you need to snap freeze with liquid nitrogen.
This post is very informative one. As all said I conclude that every enzyme have their own properties and have their own unique life spans. Therefore, after purification we have to characterized the purified enzyme by checking in which condition it will be more stable (means active). Besides temp. and pH we have to study many metals, organic solvents and other surfactants also. As all said storing temp. should be -20oC or -80oC.
My question is in liquid form can we keep our enzymes stable for 3-4 months??? Any experiences?? Other suggest me to keep enzymes in powder form which keep enzyme activity for maximum time without loosing its activity. Need suggestions...
If the enzyme is lyophilized with suitable excipients (eg sucrose as lyo protectant, buffers (citrate as buffer, chelating agent , reducing agent), it may be stable at ambient temperature for years.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology