Ethylene Glycol → 7-(Benzo(a)pyren-6-yl)guanine

Participants

Reaction Steps

While the blocking reaction is progressing, NAb (selective for BP-6-N7Gua) and BP-6-N7Gua or benzo[a]pyrene (BP) or methylguanine (Me-N7Gua) or other structures related to BP-6-N7Gua are combined in separate Eppendorf tubes (1.5 mL polypropylene tubes). MAb is diluted 1:500 to 1:5000 in TBS buffer (0.05M Tris, 0.20M NaCl, pH 7.5) containing 10% (v/v) ethylene glycol as a protein stabilizer, then 500 uL is dispensed to each Eppendorf tube seated in a thermal mixing block set at 45 degrees C. These samples are heated for 10 minutes with vortexing. BP-6-N7Gua or BP or Me-N7Gua or other structures related to BP-6-N7Gua are diluted in methanol (MtOH), then added with vortexing in 10 uL volumes to duplicate samples of heated MAb solution. MtOH, alone, is added as a control to selected Eppendorfs. The reaction mixtures are heated with vortexing for an additional 20 minutes, then the reaction mixtures are cooled by addition of 100 uL of TBS buffer with 10% ethylene glycol. The reaction mixtures are incubated an additional 45 minutes at 37 degrees C.

Provenance & Evidence