Buffer Question

I am trying to test the binding constant of a protein to cellulose; previous attempts have not produced good results (the protein was suspended in DI water) so I want to try it in buffer. How do I choose the appropriate buffer for a protein?
Thanks :)

And im new to this site so how do I post questions without creating my own topic?

what method are you using? and what the problems are? did the protein preticipitate?

you may just do not have the right concentration, what the Molar concentration of your samples? if the estimated binding ratio is 1:1, you will need a molar ration >3:1

you should be able to use Tris or PBS buffer at desired pH. Avoid DTT.

good luck

Hi dmm520,

Creating your own topic is the way to post a question. I hope qinglongyanyuedao's advice is helpful. Come back with any problems you may have, and tell your friends.

Omai

Thanks for the replies!

I'm new at protein research and prepared Tris buffer solutions at various pHs...how do I know what pH i should keep the protein at? (its pH is 4.6)

I'm injecting protein+buffer into a solution of cellulose in a calorimeter....we thought we would see binding results but nothing yet..

Do you know the pH of the native environment for this cellulose/protein interaction - balance your buffer to that pH if you do.

This is an OLD paper that looks at cellulose binding

http://jb.asm.org/cgi/reprint/175/18/5762.pdf

you might be able to lift a method maybe? This is not my field but doin' my best to help!

if your proteins work with DI H2O, I will suggest a pH at 6 or around.

Are you using the Isothermal Titration Calorimeter?

TIPS for working with a calorimeter

1. You will first need to calculate your protein concentration, since you do not have signal, seems to me that most probably that you do not have enough concentrated protein in your reaction. So, first, make sure that you have the right concentration ratio of both your protein and cellulose according to the binding constant. If you have no idea of your system’s binding constant, suggestions for starting concentrations of protein and ligand solutions are given in Section 6.5 of the VP-ITC User’s Manual. (available free online at www.microcalorimetry.com, or just PM me, I should be able to email you a PDF version)

2. and it is crucial that Protein and ligand solutions need to be in the same buffer so no heat of dilution will be generated when they mix. DTT and TCEP should be avoided in your buffer. See Section 6.3 of the User’s Manual for reasons and possible substitutes.

3. and for calorimeter, your solutions need to be well degassed first since air coming out of solution will also introduce spurious heat.

yes, there are really some tricks you need to play with when working with a calorimeter (especially the sample loading), just be patient and play with them, have fun, and good things will happen. Have fun

Let me know if have any other questions concerning with the calorimeter.
Good Luck.

Thank you for the help; I am getting binding results but instead of a digression of nice exothermic spikes, i am getting spikes above and below the baseline (i guess meaning endothermic and exothermic reactions are occurring). since the ITC machine is new in our lab the only thing we can think that would give these kind of results is that the cellulose is microbially contaminated...any thoughts? (both cellulose and protein are in DI water right now...we haven't tried the buffer yet)

it looks like you have bubbles in your system, most probably in the cell.
to avoid, first thing to do is have your samples de-gased for at least 10min. When doing the injection, first let the end of your Hamilton syringe reach the bottom of the cell, then pull it up a little very gently, and start the injection very slowly.
Always has more than necessary amount in your syringe, and do not try to squeeze the last drop out of it.

thank you so much for all the ITC help...we got good binding results!
Another quick question: can someone explain the "heat of dilution" concept to me? Should it be equal to zero in ideal conditions?

Thanks :)

below is the answers.com defination of heat of dilution



(physical chemistry) The increase in enthalpy accompanying the addition of a specified amount of solvent to a solution of constant pressure. Also known as integral heat of dilution; total heat of dilution. The increase in enthalpy when an infinitesimal amount of solvent is added to a solution at constant pressure. Also known as differential heat of dilution.

hi, can any one help me plese, i am trying to find defination for the heat of dilution. thnx

Go simply, heat dilution is

"the heat relaeased or absorbed when dilute liquids"