I am doing western. I prepared sample buffer using .5 M TRIS which was diluted from 2.5 M TRIS buffer stock having ph 6.8, I did not check the ph of .5 M TRIS and made the sample buffer using this TRIS . later on , after making the samples , I checked the PH of that .5M TRIS, It was near about 8. will this affect my sample or further process as for this buffer recommended ph is 6.8? And in one protocol, it was written that after making sample buffer adjust ph to 6.8. How can it be maintained as sample buffer contain glycerol, so it will be difficult to check ph with ph meter and it cannot be checked with ph strip too as it is already colored. so how to check. plz do reply. thanks
I suspect the problem is with your pH meter rather than your water. Clean your pH probe (see manufacturers recommendations) and refill with saturated KCl. Make sure there is a layer of crystalline KCl at the bottom of the KCl solution inside your probe, then you will know that the KCl solution is still saturated. Calibrate your pH probe using reliable standards which have not been open for too long, as pH of standards will drift over time.
Hello Lalita,
If you will use sample buffer containing tris of pH of 8, it will create problems in stacking of the proteins during PAGE run because pH of resolving gel is also 8.8 . As a result the band pattern on the blot will be distorted. It will be better to make a new loading buffer. We at our lab make loading buffer by taking 6.25 ml of 1M tris buffer of pH 6.8(buffer which is used to make stacking gel), adding a pinch of bromophenol blue to it and adjusting final volume to 12.5 ml by adding glycerol.
Hope this may be of some help to it.
I have diluted several buffers from more concentrated stocks into lower concentraion ones but never saw such a big chnage in pH. Tris is a very commonly used buffer and it will be hard to imagine pH changed fro 6.8 to 8.0 simply by adding H2O (a no degassed one may have pH 6.1-6.2 due to CO2). Make sure your containers have been cleaned before use and the pH meter has adequate amount of saturated KCl in it (a almost dried probe has fooled me several times).
Thank u so much for ur answer. Actualy i checked the ph of water which i uesd for dilution. it was about 8.5.i think due to that the ph raised that much
Wait, are you using non-DD-H2O for dilution? I is hard to imagine pure water has pH 8.5 and is stronger than the Tris buffering capacity.
I suspect the problem is with your pH meter rather than your water. Clean your pH probe (see manufacturers recommendations) and refill with saturated KCl. Make sure there is a layer of crystalline KCl at the bottom of the KCl solution inside your probe, then you will know that the KCl solution is still saturated. Calibrate your pH probe using reliable standards which have not been open for too long, as pH of standards will drift over time.
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