Dimer bands under reducing conditions - (Feb/06/2008 )
We have recently had some funny bands on Western Blots under reducing conditions (boiled samples, DTT and SDS-page gel). The sample is HA-tagged recombinant chemokine, which has been purified on an HA column, and bands are in sizes that could match monomers, dimers and trimers. Chemokines are known to have a strong tendency for multimerisation, but surely these would not be covalent and should be destroyed under the described conditions..?? Any good ideas? The bands are detected both by antibodies to the chemokine and by antibodies to the HA-tag...
Would be extremely grateful for some genius feed-back! Thanks in advance!
What is the pH of the solutions? DTT has a reduced half-life above pH8.