Hi Anamika, - Have you double-checked that the sequence of your cDNA is correct and includes the biotin acceptor peptide tag (BAP tag aka AviTag; GLNDIFEAQKIEWHE in single-letter amino acid code)? - Are you using a dedicated bacterial strain that over-expresses BirA enzyme? This may not be strictly necessary because we often find that native E. coli BirA levels are sufficient to incorporate biotin, albeit with low efficiency. - You could simply do a Western blot to confirm biotin incorporation. I like the high-sensitivity streptavidin-HRP conjugate from Pierce (cat. no. 21130).
Best wishes, Jonathan ----------------------------------------------------------------- Jonathan Elegheert, PhD Team Leader Interdisciplinary Institute for NeuroScience (IINS) UMR5297 CNRS / UB University of Bordeaux Centre Broca Nouvelle-Aquitaine 146 rue Léo Saignat 33076 Bordeaux Cedex France E-mail: jonathan.eleghe...@u-bordeaux.fr <mailto:jonathan.eleghe...@u-bordeaux.fr> ----------------------------------------------------------------- > On 13 Nov 2018, at 10:13, Anamika Singh <anamika.ii...@gmail.com> wrote: > > 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 > <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
