You don’t say so, but one assumes that you have a BAP tag on the protein, and co-express a biotin ligase such as BirA?
Ed T.A.Edwards Ph.D. Associate Professor of Biochemistry Deputy Head of School _____________________________________ Astbury Centre for Structural Molecular Biology School of Molecular and Cellular Biology Faculty of Biological Sciences ======================= Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 Faculty Staff Profile<https://biologicalsciences.leeds.ac.uk/molecular-and-cellular-biology/staff/61/thomas-a-edwards-ph-d-> Astbury Centre Web Page<http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE> Invention, my dear friends, is 93% perspiration, 6% electricity, 4% evaporation, and 2% butterscotch ripple. ~Willy Wonka [signature_1229172001]<https://poppi.website/> Perturbation of Protein-Protein Interactions From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Anamika Singh <anamika.ii...@gmail.com> Reply-To: Anamika Singh <anamika.ii...@gmail.com> Date: Tuesday, 13 November 2018 at 10:15 To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] OFF TOPIC Hi All, I am purifying the biotinylated protein (cloned into the pET28a vector) using Avidin beads. Since I need the protein for SPR but when I used the purified protein to interact with Streptavidin coated onto the SPR chip. There was no signal. Can anybody tell me why is it so or how can I make sure that the purified protein is biotinylated enough to interact and give the signal? Because when I ran the SDS-PAGE there was a band of purified protein. I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM Biotin). PS: I have included the biotin during overexpression of the protein also. Please suggest. Thanks -- Anamika Post-Doctoral Fellow Silberman Institute of Life Sciences Hebrew University of Jerusalem, Israel ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
