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SDS PAGE sample preparation question, and discussion?

electrophoresis SDS PAGE sample preparation

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3 replies to this topic

#1 kyri_osity

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Posted 22 July 2014 - 02:36 PM

If it is necessary to dilute out an SDS-PAGE sample(s) after adding the protein (to a specific final concentration), the SDS Sample Loading Buffer (final = 1x), why use anything other than pure water?  Each sample, depending on the initial concentration of the protein, could contain different volumes of the dilutent. 

notebook excerpt - SDS PAGE sample prep final concentrations.jpg  

 

If using a buffer, then each sample could have slightly different concentrations from one another.  Why introduce more than one variable in ]between samples?

notebook excerpt - SDS PAGE sample prep.jpg



#2 mdfenko

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Posted 23 July 2014 - 03:44 AM

if you use the buffer in which your protein sample is residing then you will not have different concentrations of the buffer salts, only different amounts of the protein (as you want).

 

if you use water to dilute the various samples then the concentration of the buffer components of the medium in which your protein is prepared will vary. this may have a profound effect on the results.


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#3 kyri_osity

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Posted 23 July 2014 - 08:06 AM

Using the same buffer that the protein was isolated/purified in, that I can see the logic there. Good point.
What about different buffers, such as in the example I attached in my original post (if the attachments are too poor of a quality to see the column headings, I apologize) where the individual used 1x SDS electrophoresis buffer? Wouldn't that also have similar issues, as in using water?

#4 mdfenko

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Posted 24 July 2014 - 10:46 AM

using 1x sds buffer will not equalize buffer components between samples. it may also interfere with stacking.


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