I'm trying to stabilize my protein by use of different buffers. It obviously loves alkaline pH, because below pH 6 the activity was gone in few days even when stored at -80°C. However, even at pH 8 and stored at -80°C the activity decreases (it's little weird, because until day 3 it stays at about 100% and on day 5 it drops down). However, I wanted to try other buffers, eventually with higher pH. For pH 9 I want to use Tris with either HCl or citric acid. I was thinking also about pH 10 with borate buffer, but I found out, that borate can be used only up to pH 9.2 (see the link attached). What I found was either bicarbonate-tetraborate or glycine-NaOH. Which one do you think would be better?
http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/
Hey,
Are you sure that your protein behaves well with the said pH? I would suggest you to use ProtParam tool from ExPasy (online) and find out about your protein parameters using the sequence data. You can find out what is the theoretical pI (iso-electric point) for your protein and then store them in buffers with pH farther than your pI. Your protein will be least soluble if the pH of your buffer is close to pI value.
I believe this factor is important.
Best Regards,
Sagar Sridhara
I will suggest go for glycine-NaOH buffer as it did not allow to reduce activity of protein at least 1 week. I am sure of that.
Hi,
I can´t suggest a buffer but have you tried adding Glycerol to your buffer? The addition of about 25% (v/v) or up to 50% did help a lot with my enzyme.
@Sagar Sridhara I don't know the sequence. The enzyme is brand new and I will try to purify and identify it if I will be able to keep it alive at least for a week.
@Christoph Loderer when I measured pH optimum, it was at least 9.5.
As I wrote above, pH 4 and 5 didn't keep any activity and 6 was little better, but still pretty bad in comparison to 7 and 8. For that reason I want to try at least pH 9 and preferably also 10.
Freezing the enzyme could be the reason you are loosing activity. Try Cristoph's suggestion of adding glycerol (or ethylene glycol) to your enzyme so that won't form ice crystals at -80oC. You can do this in any buffer you want.
I tried room temperature, +4°C, -20°C and -80°C with 20% glycerol and nothing helped.
Hi Tomas,
Are you sure that the activity of your protein decreases when stored at -80°C ? Normally it should be stable provided you added cryoprotectant (at least 5% Glycerol).
Try to find a good buffer (attached file can give you ideas), then try different ways of storage...
-20°C with 50% Glycerol is often good for biochemistry, notably if you dilute you stock (and thereby Glycerol) for your assays. If you make this, make sure to pipet your stock at -20°C using a cold rack for your tubes (as for most of commercial enzymes...).
Flash Freezing in liquid nitrogen (avoids denaturation due to ice crystals) and storage at -80°C is often very good too... The smallest the volume of your sample, the better (split your stock if necessary)... If you do this, also try different ways of thawing : usually thawing rapidly with the tube in your hands is the best, but some proteins prefer to be thawed slowly on ice.
Hope this will help
Sorry, I forgot to mention that flash freezing and storage to -80°C is also likely to be more successful if you add at least 5% Glycerol
not that I would measure the protease activity somehow, but I'm adding EDTA, benzamidin and bestatin (plus PMSF after extraction)
Do you know if you are losing protein, losing activity or both?
You may refer to Rex M.C. Dawson et al. "Data for Biochemical research", this book has all list of buffers you might want to try, but what i saw at your pH range, Clark-Labs buffer should be fine [Bower, Bates, J. Res. Natn. Bur. Stand. 55, 197, 1955] it has mixture of 0,1 KCl, 0,1M H3BO3 and 0.1M NaOH and good at range of pH 8,0-10,2
Glycine-NaOH is gootd at pH 8,6-10,6, and Carbonate-Bicargonate is good at pH 9.2-10.8
Tris Hcl, sodium acetate and sodium phosphate buffers are suggested for this case
I've had good luck with Tris-HCl buffers for alkaline phosphatase, but that was only stored at pH=8.5. If you need a high pH, then you might need something else.
As previously mentioned try storage with glycerol (25-50%). I have seen success in storage of my protein with 50% glycerol at -20˚C for more than a month.
Tris-HCl at pH 7-8 was appropriate for my storage conditions. I have to mention that my protein is very unstable when stored at RT, 4˚C or even at -20˚C with many other conditions.
Also to mention, freezing and thawing constantly would also denature and destabilise proteins. Hence, proteins with glycerol are recommended to be stored at -20˚C in a liquid state or as use and throw aliquots at -80˚C.
The thing is, that I need to work with the enzyme to purify and identify it, so the storage is actually not that crucial. Also, freezing-thawing seems to have no effect on my protein.
However, I just got results from my most recent measurenment and glycine-NaOH pH 10 kept 80% activity after 10 days even when stored at +4°C, so it seems promising.
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