Should DTT be added to the SDS Sample Buffer for SDS PAGE analysis?
DTT should be added to an aliquot of 3X SDS Sample Buffer to a final concentration of 40 mM. For analysis of IMPACT purification you may use SDS Sample Buffer containing DTT. However, boiling in DTT-containing SDS Sample Buffer can cause partial or complete cleavage of the fusion protein, resulting in an overestimation of in vivo cleavage. If substantial in vivo cleavage is observed, the cell extract should be evaluated in a SDS Sample Buffer without DTT. TCEP [tris-(2-carobxyethyl) phosphine] or TCCP [tris-(2-cyanoethyl) phosphine] (1-5 mM) may also be used as a reducing agent in SDS-PAGE Sample Buffer in place of DTT. TCEP is easier than TCCP to dissolve. Dissolve TCCP or TCEP in the buffer at the concentration recommended and adjust the pH to 8.5. TCEP: We used the chemical from PIERCE Catalog #20490.
