A stopped-flow apparatus for photoaffinity labeling studies in the milliseconds time range. Application in investigations of the nicotinic acetylcholine receptor.
A photoaffinity labeling method is described to label a protein covalently in various transient covalent states. The method uses a combination of an especially adapted stopped-flow apparatus with a Q-switched Nd: YAG DCR-2A laser (wavelength 266 nm, i.e. four-fold the primary frequency, pulse duration 4 ns, pulse energy 15 mJ). The construction of the mixing cell, the triggering device and the set-up for determining the dead time of the stopped-flow apparatus is described. The dead time is 2.4 ms. In combination with a specific photolabel the method has been used for labeling functional states (resting, activated, desensitized, antagonist-blocked) of the nicotinic acetylcholine receptor from Torpedo marmorata electric tissue.
D000345:Affinity Labels; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D009861:Onium Compounds; D010777:Photochemistry / Q000295:instrumentation* / Q000379:methods; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013997:Time Factors; D014101:Torpedo; D014320:Triphenylmethyl Compounds
D000345:Affinity Labels; D002038:Bungarotoxins; D009861:Onium Compounds; D011978:Receptors, Nicotinic; D014320:Triphenylmethyl Compounds; C011561:triphenylmethylphosphonium