Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

BOCILLIN FL fluorescence inhibition assay.

GK Gaurav Kumar
CG Christos Galanis
HB Hunter R. Batchelder
CT Craig A. Townsend
GL Gyanu Lamichhane
request Request a Protocol
ask Ask a question
Favorite

BOCILLIN FL (Thermo Fisher Scientific; no. B13233) is a fluorescent penicillin (λmax = 504 nm) (44). BOCILLIN FL was used at 50 μM in Tris-Cl, pH 7.4, and incubated with a fixed amount of each protein (∼200 pmol). β-lactams were used in a gradient concentration range 0 to 4,000 μM (Table S3). Each protein was incubated with a β-lactam at 25°C for 30 min, followed by BOCILLIN FL addition. The reaction mixture was incubated for an additional 30 min, 25°C, in the dark, followed by reaction quenching with Laemmli buffer and heat denaturation at 95°C, 5 min. This mixture was electrophoresed on 14% SDS-PAGE, and BOCILLIN fluorescence was imaged using a GelDoc imager (Bio-Rad). The fluorescence intensity of BOCILLIN FL in each protein band was quantified using ImageJ. Next, the SDS-PAGE gel was stained with Coomassie brilliant blue dye to verify the presence and stability of each protein.

Copyright and License information:  ©2022 Kumar et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A