We ordered a recombinant protein from a famous biotech company. This recombinant protein penetrates a particular organ and we wanted to test if that molecular process occurs in this non-model organism, it has a GFP tag. The concentration is 5 mg/mL and the storage buffer of this protein is 100 mM Tris-HCl, 50 mM EDTA and 10% Glycerol (-80 C). I am planning to inject this protein into live animals. However, the concentration recommended in other animals is 1ug/mL. Is it possible to dilute this protein with Nucelase Free water or is there any specific buffer for the dilution of recombinant proteins? I have read some papers that a saline solution or the NEBr 3.1 buffer have been used, but I am not whether nuclease free water could be another option. In addition, I would like to know whether or not the glycerol contained in the storage buffer could affect the activity of this protein during the process of injecting live animals.
I would probably dilute the protein in phosphate-buffered saline at the pH that is characteristic of the blood of the organism, probably close to 7. I don't think the residual buffer components are a problem if you are going to make a 5000-fold dilution. If the protein is going to be injected in a concentrated form, the main toxicity issue is the EDTA.
I would not use water as the diluent because then the pH could shift quite a bit and the solution would be hypotonic for the organism.
You should do the protocol that is approved by your IACUC committee. Talk with your advisor before you start anything.
Dear Jose Maria Aguilar-Camacho
as Adam B Shapiro suggest the use of a buffer as PBS is preferable than just water since the the regulation of pH and ionic strength manitain protein stability and avoid possibile aggregation.
If really you have to dilute your sample 5000 times, i will not be worried about presence of glycerol and other elements since their final solutiion will be really low
Are you sure that 1ug/ml is correct? Since in to my knowledge in general for example when you are immunizing a protein in animals as vaccines or to stimilate antibody production we use 1-2mg/ml and not ug/ml, however your intedend use is completelly different and is possible that since you have a fluorescence tag even vey low amount are enogh.
best regards
Manuele
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