What method for purifying biotinylated proteins will you utilize? I used streptavidin magnetic beads because it's easy to do a lot of samples at one time and there's a very effective protocol for eluting the proteins.

Cheah, J. S., & Yamada, S. (2017). A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat. Biochemical and biophysical research communications, 493(4), 1522-1527.

With this protocol, I used 300 ug protein in 100 uL, but I don't see why you can't use a larger volume and an even more concentrated solution. If you plan to identify the proteins by MS, then you should consider using about 1 mg of protein, otherwise, you will not see low abundant proteins. Also, carboxylases naturally contain a biotin group so you will not be able to determine if your protein interacts with those using this assay.

Thank you for the information Kendall. I was planning to do a pull down assay using Biotinylated Protein Interaction Pull-Down Kit. I was thinking if i do it with less concentration of protein, will it work ? Their suggested concentration is a minimum of 0.5 mg/ml.

Will using lesser concentration lead to poor MS profile?

I would follow their protocol because I did not use the kit. You might need to scale up the amount of biotinylated protein you use though if you do not get enough interacting proteins on your first trial assay.

Sure, Thank you Kendall.

Short answer: protein concentration doesn't matter for the pull-down per se. What matters is your ability to detect interaction events. Strept/biotin interactions are quite strong and reversing the binding is relatively difficult; protocols involving biotin coupled to denaturation (e.g. Laemmli buffers) are optimal. Would use anywhere between 0.1 to 2 mg/ml (more dilute will work, more concentrated may give you precipitation issues). Also, not sure which beads you're using but we've had issues with binding in some experiments that may be linked to some aging beads (lower efficiency/capacity); make sure you confirm depletion of your biotinylated target from the supernatant.