Atto 643 nhs ester

Actin was fluorescently labeled on the surface lysine-328, using Alexa Fluor 488, Alexa Fluor 568, or Alexa Fluor 647 NHS ester (Thermo Fisher Scientific), or ATTO 643 NHS ester (Atto-tec) as described in detail in (57 (link)). The Arp2/3 complex was fluorescently labeled using Alexa Fluor 488 or Alexa Fluor 568 C₅-maleimide (Thermo Fisher Scientific). The protein solution was prepared for labeling by performing a buffer exchange to remove dithiothreitol (DTT) from the solution. This was accomplished by passing the protein solution through a MicroBiospin 6 column (Bio-Rad). The exchange buffer contained 20 mM Hepes at pH 7.2, 0.2 mM MgCl₂, and 0.2 mM ATP. The buffer exchange was performed by centrifuging the sample at 1000g for 4 min. Next, a 10-fold excess of Alexa Fluor 488 (or Alexa Fluor 568) C₅-maleimide dissolved in dimethyl sulfoxide (DMSO) was added to the Arp2/3 complex solution and incubated on ice for 1 hour. The reaction was stopped by adding 1 mM DTT to the solution. To remove any unreacted excess dye, a MicroBiospin 6 column (Bio-Rad) was used by centrifugation at 1000g for 4 min. We obtained on average 3.5 Alexa dyes per the Arp2/3 complex.