HomeFAQsI am using Ph.D.™ Phage Display and the streptavidin control experiment did not yield the HPQ consensus sequence.
FAQ: I am using Ph.D.™ Phage Display and the streptavidin control experiment did not yield the HPQ consensus sequence.
If you used low pH glycine rather than biotin to elute your phage, you will likely not get an HPQ consensus sequence. Due to the relatively low affinity of the peptide-streptavidin interaction, nonspecific elution is incapable of selectively enriching for HPQ-containing peptides. HPQ-containing peptides can be competitively eluted using the natural ligand biotin. If you used biotin to elute and still did not get a consensus sequence, the most likely explanation is that you did not carry out sufficiently rigorous washes. When you wash, pour the wash buffer in the plate from a bottle (don't gently pipet it in) and swirl it for about 10 seconds each time. The number of phage that you elute after the first round of biopanning should be in the range of 103 - 107 (closer to 103 for an ELISA well and closer to 107 for larger wells). If you are eluting more phage, you are not washing well enough and as a result, not getting sufficient enrichment. It also may help to add 0.1 µg/ml streptavidin to the blocking buffer to complex any contaminating biotin in your BSA, which could otherwise complex the streptavidin on the plate during the blocking step.