Just curious because this seems like a common procedure but not sure why is this step necessary.
Since DTT is a reducing agent, which 'breaks' disulfide bonds, it is added to homogenisation buffer to better denature proteins (for better seperation in electrophoresis).
As for the sucrose, it's usually added to render an osmotic balance. You can find more about it in the following link:
http://www.answers.com/Q/What_is_the_function_of_sucrose_in_the_homogenization_buffer_during_fractionation_of_cells
Dear Ms Elisa, thank you for responding to my enquiry.
I am actually quite confused because DTT can also be used to stabilize enzymes and other proteins, which has these free sulfhydryl groups. So my question is, if i were to isolate an enzyme (which happens to reside on the nuclear envelope) for functionality studies, do i add DTT to my cells before homogenising them? Because some papers have reported using varied amounts of DTT added for this step before homogenisation.
For your advice, please.
Warmest regards,
Justin
In general, working with proteins from an intracellular environment means working with proteins that normally exist in a reducing environment, and DTT helps maintain that environment and keep the proteins happy. Secreted proteins, or the extracellular domains of proteins, are exposed to a more oxidizing environment, and are often held together by S-S bonds, so adding high concentrations of DTT or another reducing agent will not be good. As an example, antibodies are held together by S-S bonds, and can be stripped from blots by heating with beta-mercaptoethanol. Similarly, heating with BME is used to reduce proteins before SDS-PAGE to reduce any S-S crosslinks so the proteins can run as linear polymers coated with SDS.
Hello,
as your purpose is to do an enzymatic activity study, you need to keep your protein in its native so I would suggest not to add DTT because it will unfold your proteins and therefore will make them lose their original tri-dimensional conformation which is crucial for their function.
Cheers
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