Hello!
I am preparing a protocol for cell lysis of tissue samples. I want native conformation of as many proteins as possible and would therefore like to have good pH control.
In most protocols, I see the use of Tris-HCl, which I thought would be less suitable for cell lysis, as most lysis protocols are performed at 4°C.
Say I make the buffer at room temperature, then lyse cells at 4°C, then maybe store the lysate frozen before thawing the lysate and using it at room temp for analyses like immunoprecipitation or functional assays, wouldn't Tris give you pretty shaky control of pH?
I am considering using HEPES or PBS, but I find much fewer examples where these buffers are used in lysis buffer (especially PBS).
In general, I don't think the choice of the buffer is a major factor for cell lysis as long as the buffer has its pKa near (+/- 0.5 units) the pH and the concentration is high enough to keep the pH from drifting.
One reason for the popularity of Tris is it's low cost and common availability. I agree that if the concentration is too low, the pH may not be well-maintained. Also, the pH will shift with temperature. Its pKa of 8.1 is also a also bit high compared to physiological pH (~7.4).
Phosphate buffer is inexpensive and readily available. There is a phase transition in phosphate buffer when it freezes that releases heat, so it is not great if you are planning on freezing samples slowly at -20oC.
HEPES is a very good choice, with its pKa close to physiological pH and relatively small change in pH with temperature, but it is more expensive.
Hi Johanna Wagnerberger you are right that Tris' pKa is changing with temperature. When I knew I'm going to prepare Tris buffer, I've put water into fridge e.g. in the afternoon to be it ready in the morning for buffer preparation. Then the preparation of buffer was suffficiently quick, so I didn't bother with cooling, because the temperature didn't manage to rise so much. But if you were afraid, that the dissolution may take too long (e.g. if you use high salt concentration), you can dissolve it in advance as well and then only adjust pH.
Phosphate buffer has disadvantage that phosphate interacts with many enzymes.
Thank you for all the great help!
Adam B Shapiro I will probably snap-freeze the samples in liquid nitrogen if I freeze them, but I think I will go for HEPES anyway.
So-called "physiological" pH, meaning the pH of human blood plasma, is about 7.4. That, I presume, is why slightly alkaline buffers are so commonly used.
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