Most protocols suggest the use of DTT (Dithiothreitol), a reducing agent in the isolation QB buffer for extraction of protein from plant tissue. I plan to run an SDS-PAGE gel. Is there an alternative to DTT for extraction? Is it absolutely necessary to add DTT?
I read up that it is used for denaturing the proteins and breaking the disulfides. Since beta-mercaptoethnol does the same thing, can it act as a substitute? If yes, at what conc. in the extraction buffer?
If I'm adding glycerol and DTT/mercaptoethanol in the extraction buffer, do I need to add them again during sample preparation (95 oC boiling) for SDS PAGE loading?
Reducing agents are necessary for disulfide bond reduction and complete denaturation of protein that contain disulfide bonds.
Yes you can use beta-mercaptoethanol as an alternative to DTT if you don't have DTT.
Generally DTT is preferred because it is not volatile and therefore doesn't smell nearly as much as beta-mercaptoethanol, is effective at lower concentrations and is more stable.
You could also use TCEP.
2.5-5% mercaptoethanol or 10 - 100 mM DTT in sample loading buffer.
Reducing agents are added to protein extraction buffers at lower concentrations (0.1-1mM DTT) to prevent protein oxidation rather than disrupt disulfide bonds.
Yes add glycerol and reducing agent prior to boiling. 70 degrees for 5-10 min is generally hot enough though. Remember to cool on ice before loading.
Reducing agents are necessary for disulfide bond reduction and complete denaturation of protein that contain disulfide bonds.
Yes you can use beta-mercaptoethanol as an alternative to DTT if you don't have DTT.
Generally DTT is preferred because it is not volatile and therefore doesn't smell nearly as much as beta-mercaptoethanol, is effective at lower concentrations and is more stable.
You could also use TCEP.
2.5-5% mercaptoethanol or 10 - 100 mM DTT in sample loading buffer.
Reducing agents are added to protein extraction buffers at lower concentrations (0.1-1mM DTT) to prevent protein oxidation rather than disrupt disulfide bonds.
Yes add glycerol and reducing agent prior to boiling. 70 degrees for 5-10 min is generally hot enough though. Remember to cool on ice before loading.
I completely concur with James excellent answer; and cannot add anything else to it
You can use 2-mercaptoethanol instead of DTT. A concentration of 0.05% (v/v) is recommended for most applications.
Other alternatives are DTE or TCEP (tris(2-carboxyethyl)phosphine).
TCEP has several advantages compared to DTT or 2-mercaptoethanol:
- odorless
- more powerful reducing agent
- irreversible reducing agent
- more hydrophilic
- more resistant to oxidation in air
Regards, Christian
This comparison might help you
http://agscientific.com/blog/index.php/2011/11/17/side-by-side-comparison-dtt-vs-tcep-preferred-reducing-reagents/
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