Can any one suggest me concentration of beta-mercaptoethanol (2-ME) or DTT to prevent cysteine dimerisation in protein purification at pH 8.0 . and stability of them in Tris Hcl buffer with EDTA .
Are you planning to carry on an IMAC purification step? In this case you should not be adding a lot of DTT (1 mM but your column can turn brown), beta mercaptoethanol (up to 20 mM but that is a lot, try 5 mM). Otherwise you can try TCEP (1 mM) but it is pretty expensive. The stability in solution will depend on the pH and the temperature of your buffer. For DTT goes from 40 min. to 9-10h if kept in low temperatures.
We are planning to carry out Ion exchange chromatography , at pH 8.0 , with Tris Hcl and EDTA buffer , I wanted to know the stability of the reagents so that we can understand the stability of protein , (i.e, without dimerization)
You can use 6 mM B-ME or 1 mM DTT or 1 mM TCEP. As Jorgaq Pata pointed out never use these reducing agents if you are performing IMAC. Whereas, if you are performing IEX, there should not be any problem at the above mentioned concentrations.
Stability of these reagents is dependent on the buffer temperature. B-ME is the least stable among the three. Considering the cost factor and the stability I suggest DTT to be a better bet. DTT is generally stable upto 12 hours.
DTT at 1-2 mM or TCEP at 0,5-1 mM. For ß-ME go to 5-10 mM.
Note that EDTA will bind to anion-exhangers. From Q-sepharose it elutes at around 250 mM NaCl, so proteins eluting before that concentration will not have EDTA! Also if you over-equilibrate the column with EDTA in the buffers you will see reduced binding capacity because of accumulated EDTA.
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