Hi there,

The pulldown experiment in vivo will demonstrate that the interaction does occur in a cellular context . The other way to show an interaction in vitro would be to immobilize the first partner on the corresponding affinity column and then pass the second partner onto the column and demonstrating that they coelute when breaking down affinity of the first partner to the column. The advantage of this approach is that you can also easily test the strength of the protein protein interaction by varying the ionic strength of the wash buffers.

Hope it helps...

In vitro pull down is a valid approach, but it does not tell you whether these proteins interact in vivo.

Do not forget to include GST only control with His tagged protein, to exclude unspecific binding.

You could also do this assay in the presence of cell lysate. This will increase the competition and could be closer to the in vivo situation.

Two purified proteins could be interacting simply because there is huge quantity of them and they are not separated into different compartments in the cell, or aren't occupied by their normal interaction partners. Therefore it is more beneficial to express these proteins in the cell, to prove the interaction. It could also be that the interaction of the two is regulated by certain factors that are only present in the cell and not in your in vitro system.

Hi Howard, as Maria and Michael said, this will not be an easy thing to do.

But, if your proteins bind tightly with a Kd < 10e-8 M, then you can make a case by incubating them in a dilute solution and pull them down together.

If your proteins bind with a Kd > 10e-6 M then it will be an uphill battle to show that they are specifically binding.

If you don't know the Kd of your proteins, you can get a rough idea by immobilizing one of them onto a 96-well plate, blocking with 1% BSA and do a serial dilution of your other protein onto the wells and detect it with an antibody. 

Howard, you've gotten some fine advice here. As I just finished commenting on another co-precipitation complex problem, the suggestions of negative control and non-specific his-tag binding are huge! 

Any, yes, this will be a challenging experiment to do. It seems that you've gotten both in-vitro and in-vivo replies and I'm not sure which you are looking for. At the surface, it seems as though you are asking an analytical in-vitro question.

However...now this depends on two things...how much money you have OR how willing are you to go to the nifty Biophysical Chemistry analytical lab? Usually these kinds of facilities have a titration calorimeter...a really cool instrument which will give you the heat of binding...assuming you've done the controls as well as the binding constant extrapolated from the Heating curves. 

A second and really cool experiment...I like to 'see' things in real life...I like Maria's comment...and I would put some wheels on what she'd said...at least I think so. 

If you are a nice guy and know the right people who actually understand how to 'really use' a confocal Imaging microscope and you happen to have your two complexes labeled with a Chromophore, be it a single probe or a CFP or GFP or YFP, using the abilities of a confocal microsope to produce 'z-section cuts' of your cell of interest, you could micro-inject your major protein into the cell, identify its location based on its fluorescent signal (and this can't be done on a conventional Fluorescent microscope) and then, micro-inject your second protein at varied concentrations. Allow an equilibrium amount of time for diffusion and then look for the FRET signal. As a function of concentration, you should be able to quantify your FRET signal...it will be hard to get the low binding affinity site data but this does answer the in-vivo question of both location of your complex relative to other cellular features...which could be telling...and affinity constants for at least part of the binding curve.

Well, my friend, I am off to write a paper after finishing some reading. This has been a nice diversion for a few minutes to talk science.

I hope the comments you've received have been helpful,

David

David W. Chester, Ph.D.

BioAnalytical Consulting, LLC.

Hi everyone, thank you for your replies.

I plan to do the pulldown with the prey protein as a second recombinant pure protein, as well in total cell lysates. In the past, I have done IPs using total cell lysates at 1mg/ml with 1ml total volume.  Because I want to use a pure protein as a prey protein, I am unsure an ideal concentration to start with.  I was thinking using 0.01ug or 0.001ug  of prey protein (the His-tag protein) in the pulldown. I know if the concentration of the prey protein (purified protein) is too high, many non-specific bindings will occur.  

At the moment, I am only interested to try answer the question "does protein A bind with protein B".

I will incorporate everybody's suggestions and try more experiments once I can confirm the recombinant proteins bind to each other.

Hi Howard, I admire your commitment, but you could get a better idea of the interaction between protein A and B by coating one of the proteins onto an 96-well ELISA plate, wash and block with 5% BSA.

Then titrate your other protein into the wells (in the presence of 5% BSA, wash) and detect the bound protein with an antibody against your tag.

Which protein you coat and which protein you want to detect depends on the anti-tag antibodies you have.

You can contact me for more details about getting an "apparent Kd" for your proteins..