The short answer is YES. This would depend on several factors such as the antibody that you plan to use for IP/co-IP and the abundance of the protein of your interest in the total cell lysate. If the protein of interest is not abundant, detection of the protein in IP could be challenging. In general, one should start with maximum protein amount possible.

Hello Julie,

as already said, of course you can use less protein amount. I can imagine that with third instar larvae you need quite a few of them to collect half a mg of protein...! And don't forget that a proper IP will require a negative, or mock IP, control (so, if you exploit the GFP moiety using a GFP Ab you will need to do an IP without the Ab - using only the proteinA/G beads that are used to catch the Ab; or if you use the Chromotek GFP-trap system that employs a nanobody covalently bound to sepharose beads you will have to use sepharose beads alone; same if you will use StrepTactin beads exploiting your strep-tag).

Clearly the less starting material you use, the less you can recover/elute after IP, so in order to load enough material on the WB, you will have to try and keep low the elution volumes (i.e., if in a typical IP you would elute in 100 ul of 2x SDS sample buffer, try to go down to 50 ul... less will be difficult, because you always have some dead volume in your beads). Of course you should also try to dose the beads amount to your protein quantity (i.e., if in a typical IP 50-100 ul of slurry are suggested, you can go down to 20-50 ul). Finally, if you will be using an Ab first, normally Ab binding is more efficient in small volumes with higher protein concentration (around 2-5 mg/ml), so this will mean incubating in very small volumes, for example 50 ul... We normally rotate the tube on a wheel during this incubation, but with such a small volume it would not help, so you might want to gently shake it in a 2 ml tube that has a round bottom. If you use the GFP-trap or StrepTactin beads, you normally have two possibilities, incubate with the extract in the tube or place them on a microcolumn and pass the extract on several times. The latter is normally more efficient and cleaner, so in that case you will need to dilute your proteins to have at least 200 ul, and pass the extract at least 5 times to make sure you bind all possible protein. Anyway save and check the flow through by WB to see how efficient was your IP/pull-down (in fact with the strep-tag/StrepTactin system is technically more a pull-down than an IP), loading the same volume/amount you will load for the input fraction: you should not see much of your protein, if the experiment worked well, maybe nothing, or a lot less than in the input.

Good luck!

The amount of protein you want to use for IP depends on they bait expression level, the prey's affinity and sensitivity of the antibodies you are using or the IP and western blotting. Given you have Streptavidin and GFP tag it is good because you can use Streptavidin beads or GFP-Trap beads to capture your protein. I generally go with 1mg protein to start and work my way from there. 

Ok thanks to all of you. I will try it :-). It is the best way to learn.

All the best,

Julie

Thank you all.

I was able to pull down lipin from 3rd instar larvae. :-) :-)