Question about Tris-Hcl and PBS buffer - (Jan/22/2007 )
Maybe it is a very stupid question..
but i would like to ask what is usage of buffer...
and what is the difference between the Tris-Hcl and PBS...
How can i choose buffer for the experiment? What is the basis of selecting a buffer solution?
Remember that biological reactions work well only in a very narrow range of pH (around 7.4). Many of that ractions generate or consume protons, so a buffer must have the capability of compensate that variations of pH. Tris has pros and cons about it. Its pKa is 8.0, so its buffering capacity is around 7.5-9.0, enough to most of biological reactions. Despite its high buffering capacity, being highly soluble in water and being inert for enzimatic reactions, Tris is a toxic polyamine, and that's why we use PBS for living cells experiments. PBS is made of phosphates so it's cheap and easy to prepare (an important point for a good buffer). It's no toxic for cells, compatible in osmolarity and it's insensitive to temperature changes (Tris don't). Phosphates have a high buffering capacity too but may inhibit enzymatic reactions because they can sequester divalent cations like calcium and magnesium, then we use Tris for enzymatic reactions.
Choosing a buffer depends upon the objective of your experiment and the nature of your method (enzymatic reactions, histological procedure, live/dead cell detection methods, etc). Take a look on the literature about the experiment you want to do and check is buffers used there are compatible with your experiment or protocol. Some basic books about cell culture or molecular biology technics are useful too. Even brochures or catalogs from manufacturers could be a good source of information.
Phosphates have a high buffering capacity too but may inhibit enzymatic reactions because they can sequester divalent cations like calcium and magnesium, then we use Tris for enzymatic reactions.
Well you learn something new every day, thanks Aleruiz
Thanks for your kind reply and detailed explanation, Aleruiz.
Thank you aleruiz for the explanation of Tris and PBS.
thank you also....you have already helped me alot