Hi all,
Can one use Li Lauril Sulfate (LDS) in loading buffer to run protein samples containing high concentrations of KCl, on SDS-PAGE? Does one need to replace SDS in the running buffer and in the gel as well? LDS is much more costly than SDS, thus, it would be great if one could get away using LDS just for the loading buffer.
Thank you in advance!
To my understanding, you are going to get massive precipitation of Potassium Dodecyl Sulfate when mixing your samples with the loading buffer regardless if the latest contains Lithium-DS or Sodium-DS. Afterwards, it doesn't really matter whether you have Lithium-DS or Sodium-DS in the gel and running buffer.
If you want to remove KCl from your samples prior mixing with the loading buffer, you may try adding to them small volume of concentrated NaClO4 or LiCl4. Potassium Perchlorate has relatively low solubility in aqueous solutions, and will precipitate out while leaving behind NaCl or LiCl, respectively. The precipitate is dense and can be easily removed by centrifugation. This way, you can decrease the depletion of Dodecyl Sulfate when mixing the pre-treated samples with the loading buffer.
In general, you want the concentration of extraneous ions in electrophoresis to be as low as reasonably achievable (certainly less than 50 mM), to lower Joulean heating and to prevent band distortion. IMHO the easiest and most reliable way is to precipitate the protein with MeOH/CHCl3 (doi:10.1016/0003-2697(84)90782-6).
LiDS is more soluble than SDS and hence used for acidic gels or at low temperatures.
Thank you, Victor and Engelbert. I see the point of keeping the salt (any salt) low, but, in general, when I load NaCl-containing samples (even if NaCl is 500 mM or higher) the gels look acceptable. These are 'quick tests' to identify positive fractions coming out of FPLC, so I am trying to minimize additional processing to save time. Regarding precipitating proteins: we work with quite dilute proteins, and often detect them with fluorescence rather than with Coomassie, amounts can be as low as 1 ng per lane. Thus, I am worried to lose them in precipitation.
The trick of removing K+ from the solution sounds interesting, I will try that! Centrifugation is not a big deal in terms of processing time.
When the MeOH/CHCl3 precpitation is properly performed, proteins form a thin film at the interface between organic and aqueous phase, which is easy to isolate. Thus, yield is high even with dilute samples. For very dilute samples, the precipitation can be used even to concentrate, to get enough material in a small enough volume for electrophoresis.
Englebert, Alright, I will give it at try, following your link. I suppose, I can remove the organic phase, and then remove as much as I can of the aq phase (to reduce the amount of salt), then bring the volume back with SDS loading dye?
As described in the paper, the organic phase is CHCl3, it is diluted with MeOH so that its density is reduced and the protein film can be spun down.
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