Hi All,
I am trying to use a technique used by previous graduate students that involves growing yeast in SC media -Uracil buffered to pH 6.75. All the literature published from our lab states that the growth media was buffered to pH 6.75 with 100 mM HEPES.
My problem is that I have tried multiple times to buffer the solution and have not gotten a stable pH through the growth cycle.
Initially, I prepared 1M HEPES buffer pH 6.75 and diluted it 10X to reach 1X media concentration, however upon dilution to 1x, the pH drops significantly. I then prepared the 1X media with the 100 mM HEPES, and adjusted the pH with NaOH and KOH (two separate attempts), however the pH was not stable through during growth and acidified significantly. I then buffered the 1X media with a pH 6.5 and pH 7.5 100 mM HEPES buffer, and the same issues persisted.
Does anyone have any insight into what I an missing here?
Dear Darius Chernitsky
If you culture yeast in SC media and study the growth, the pH of the media will not be constant, it will be acidified due to fermentation. The cytosol will be acidified and the cell will pump out the excess proton to the media, therefore, the media pH will be reduced significantly.
Satyendra,
I understand that, but the media is buffered to maximize the mebrane potential by collapsing the internal and external pH gradient. Do you have any insight on why a 100 mM HEPES solution would not holding its pH even slightly (acidification to pH 4.5/5, which is the same as unbuffered growth media)
The pKa of HEPES is 7.3 at 37oC. This means that if you start at a pH of 6.75, the ability of the buffer to resist acidification is compromised. Buffering with HEPES at pH 6.75 would be a good idea if you were trying to resist an increase in pH.
You could start with HEPES buffered above its pKa, such as 7.5, to reduce the acidification. Or, you could use a buffer with a lower pKa, such as MES.
Hi Phil,
It is an ongoing project so I am being a little vague on the details, but the gist of the gradient collapse is that in cerrevisea, you can mimick a high membrane potential for studying membrane protiens. The protien of interest functions naturally at a very high DeltaPsi, which is why we are mimicking the conditions. I didn't mention this in the main question, but a high extracelluar concentration of K+ is also required.
The choice of buffer is one of the things being investigated, previous researchers utilized HEPES with good results, so the question was mainly aimed at trying to recapitulate the same phenotype seen.
However, I believe now it was not the buffer identity, rather the cell lines were the major issue.
Hope this sheds some light!
Apologies,
I am on mobile as I am out of the lab, so the spell check is lacking.
We utilize a drug which is toxic to S. c. and is transported by our protein. It is expressed in the plasma membrane and its topology is such that it transports the drug into the cell, leading to a growth delay in inducing media that is proportional to the isoforms ability to transport the drug. (We occasionally pair this with tritiated drug uptake and the correlation is actually quite striking. I can send the references to you privately if researchgate has the functionality.)
The transport is highly dependent on the membrane potential, so when working at a low concentration of the drug, no isoform distinctions are seen without an increase in membrane potential.
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