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Importance of Reducing Agent


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#1 jiro_killua

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Posted 02 April 2009 - 08:07 AM

Some time ago I borrowed a loading dye from a lab when I aliquot my protein samples

Later, when I do western blot, all my target bands did not show up where I expect them to, in most cases, the apparent band size is higher than I expect

Now, I remember I couldn't "smell" any reducing agent in the loading dye I borrowed....


My theory is that the absence of loading dye would really affect the protein size in SDS-PAGE

Am I correct?

#2 Dr Teeth

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Posted 02 April 2009 - 09:16 AM

Some time ago I borrowed a loading dye from a lab when I aliquot my protein samples

Later, when I do western blot, all my target bands did not show up where I expect them to, in most cases, the apparent band size is higher than I expect

Now, I remember I couldn't "smell" any reducing agent in the loading dye I borrowed....


My theory is that the absence of loading dye would really affect the protein size in SDS-PAGE

Am I correct?


SDS and boiling already denatures proteins primary and secondary structure. Reducing agents like DTT or BME are only important for proteins that possess disulfide bonds that contribute to tertiary or quaternary structure as these agents disrupt these bonds. Most proteins won't be affected by the presence or absence of DTT or BME in your loading dye, but others will. It depends on your protein of interest.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 Lusen

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Posted 02 April 2009 - 09:52 AM

Some time ago I borrowed a loading dye from a lab when I aliquot my protein samples

Later, when I do western blot, all my target bands did not show up where I expect them to, in most cases, the apparent band size is higher than I expect

Now, I remember I couldn't "smell" any reducing agent in the loading dye I borrowed....


My theory is that the absence of loading dye would really affect the protein size in SDS-PAGE

Am I correct?


The reducing agent I buy from Invitrogen (Novex gels) hardly smell at all. Possible that it did contain reducing anyways




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