I am doing a wet Western transfer and yesterday used Transfer buffer (1X Tris-Glycine) and today did the same exact Western transfer protocol but with 0.1% SDS Transfer buffer. Yesterday, I ran it at 100 V for 1 hour and the current was around 130-140 mA. Today, with the SDS Transfer buffer, the amperage went up to 150 mA and the voltage only was at 54 V (the max current on the power pack is at 150 mA). Could addition of the SDS have added that much of an ionic strength that made the current increase so much? Didn't know how significant it was to the transfer.
0.1% SDS is only about 3.5 mM, or 7 mM ions. This doesn't seem to be enough to appreciably increase the ionic strength of the buffer.
Were you able to measure the conductivity, mS/cm, of the buffer at the run temperature to see the effect of temperature and confirm the SDS concentration? For the voltage to go down by half while increasing current by ~10% it seems the resistance would need to decrease significantly with 0.1% SDS. Was there any difference in the gel between the two runs? Even though the SDS is below the CMC and is a single charge polyatomic anion with a monoatomic single charge counter ion, the ionic activity would need to be significantly higher unless there is some ionic contamination. It may not have adversely affected the protein chemistry as with organic solvent-modified buffers or the amount transferred and might have even benefited transfer at the higher MW.
As Adam indicated,adding 0.1% SDS will only add about 7 mM of ions to the buffer. At the pH of a trie-glycine buffer (tris 25 mM, glycine 192 mM) glycine will be fully charged and tris will be about 50% ionized, so they will be contributing almost 210 mM of ions. On another note, when we perform a transfer in a BioRad minigel transfer cell (generation 1) using a trie-glycine buffer at 100 V (constant voltage) the starting current is around 350 mA. The parameters of your second transfer sound correct. (Note, the transfer is following V=IR)
Thank you all very much, these answers are helpful. I guess the main issue is that the power pack does not go above 150 mA, everywhere I have looked the power packs usually go well above that amperage. I will likely just run the transfers at max amperage instead of using constant voltage.
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