Due to higher salt concentraion the specific protein can aggregateed?
Dear Dan.B
when the chromatographic eluted specific protein was eluted with higher salt concentration, after immunization to animals It can elicit immune response, if No whether the higher sodium chloride can not support the immune system of the animals?
In general, it is a good idea to dialyze out the salt if it is way above the physiological concentrations.
Hai Scheherazade Sadegh- Nasseri
During salting out via dialysis ,protein loss is more, the proteins are permeate along with salt.
DO you know of the size of your protein? Use dialysis tubing with pore sizes smaller than your protein and you'll be fine. Please read the information in the link below: http://www.spectrumlabs.com/dialysis/FAQ.html
Good luck.
my protein size range is around 66 KDA , i am using with 15 KDa dialysis tubes.
Is your protein a multimeric protein? Or, perhaps it is not pure and the contaminants of smaller size dialyze out. Run a SDS-PAGE to verify the purity of your protein after purification. What protein are you purifying?
It iis a rabies viral Glycoprotein, i checked with SDS- asingle prominent band it showed.
Despite a prominant single band if there are smaller size bands as well, they may dialyze out- and that is good as you do not want to make ab against contaminating proteins, only the major band. In the presence of multiple proteins as immunogen, a hierarcheal competition can occur. A silver stain can better stain contaminating proteins.
Yes, first i done the SDS PAGE with coomasive blue staining, then silver staining.,Ho can i diallyse out he other contaminaint protein.
If only they are smaller than your MW cut off dialysis tube they would dialyze out. Refer to th e link http://www.spectrumlabs.com/dialysis/FAQ.html for choosing the right pore size
That depends a lot on the specific protein and its concentration. Surely there are some proteins that would aggregate when the salt concentrations increases. But just very broadly, compared to pH and maybe very low salt, higher salt concentrations are maybe not the most critical for most average proteins. But again it depends a lot on your specific protein. Also depends on how high your salt concentration was.
Any method to assess the conformation of you eluted protein like gelfiltration, native PAGE, CD or antibodies to conformational epitopes would be very good to include. Also you can maybe find some literature or at least indications on the stability of the protein (has it been produced recombinantly? Is that easy? and so on..)
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