If you have candidate binding partners, you could certain try to overexpress both proteins to increase the likelihood of detecting an interaction. However, if interactions are weak or transient, you should consider the following strategies:

1) maximize the efficiency of your IP. Usually this can be done by using antibody that is pre-conjugated (covalently cross-linked) to beads, and by using epitope tagged proteins.

2) consider using in vivo, reversible cross-linking agents such as DSP to trap your complex. If you have any insights into regulatory events that might affect protein-protein interactions, you those to your advantage. For example, treat with phosphatase inhibitors such as sodium orthovanadate if the interaction is phosphorylation dependent, or treat with an agonist/stimulant for a signal transduction pathway that might upregulate these interactions

3) use another strategy for detecting weak protein-protein interactions such as yeast two-hybrid. Often not applicable to membrane proteins, but if you are looking at cytoplasmic domains of membrane proteins, this might work with appropriate domains.

Hi Miao,

I have some difficulties understanding your issues. I think you should clarify your thread so as to receive better feeds.

1/ If the ligand for your receptor is unknown how can anyone predict it is low affinity?

2/ Normally a ligand is a small molecule that becomes “swallowed” by the receptor not an interacting partner. If you think your receptor is low affinity then you can’t over-express the receptor. You need to increase the concentration of the ligand not of the receptor and have an assay that can monitor its binding. Because your receptor is membrane-bound so I am not sure you can do a CoIP regardless of the nature of the ligand.

3/ If you still think that it is a ligand than as Daniel said you can carry out a candidate approach provided you have an assay to measure its binding. There are several ways to achieve that but again I have no experience treating with receptor-bound membrane. Maybe you could try to have only the extracellular part of your receptor to first identify the ligand and proceed for more comprehensive study.

Thanks Daniel and Abdelhalim, I think you both give me some helpful suggestions.

Let me answer the Abdelhalim 1/ quetions: some scientists connect fluorescence

to molecular X, and found the membrane of other cells which contact with it is full of fluorescence. So is the hypothesis comes out that the ligands is on the membrane. But our lab have tried many times that the co-IP assay is not useful to pull down the ligand.

Can I ask another question to Abdelhalim?

" If you think your receptor is low affinity then you can’t over-express the receptor. You need to increase the concentration of the ligand not of the receptor and have an assay that can monitor its binding."

Why I can not overexpress both the receptor and ligand? If I want to overexpress the ligand but matain the same receptor as you said , what should I do?

Can both Daniel and Abdelhalim help me more about this assay, that I want to know indead what is a ligand of a receptor? I am new in this area, and I should learn more about the protein interaction assay. Can both of you give me more advises? I am very grateful for that!

Thank you.

Miao- thanks for posting this additional information. It changes the picture completely. My initial understanding was that you were looking for protein-protein interactions within the same cell. Now it seems instead that you are looking at cell-cell interactions in the mold of something like Notch-Delta signaling.

Ligand-receptor interactions across cells often involve complicated conformational changes in these proteins, and in some circumstances may require actively applied forces (membrane tension or cell-cell tugging forces) to maintain the ligand-receptor interaction. In such circumstances, you will not be able to reconstitute the interaction in a cell-free system (that is upon cell disruption in an IP).

You may need to use live-cell methods to identify this interaction. What was the fluorescence assay used by other labs?

Hi Miao,

It seems that Daniel may be better qualified to technically help you on this specific issue. I will respond to some of questions.

By definition a low affinity receptor will only interact with the ligand when the later is present at high concentration. (High Km). It doesn't make sense to overexpress the receptor or both.

As I said, a ligand is a small molecule so it can be a hormone or a chemical compound. It is not normal to have a low affinity receptor for a hormone, growth factor, etc…. Anyway, to over-express the ligand you have first to identify it. For this, I think you should carry a candidate approach.

Is the fluorescence you are seen intrinsic to the receptor?

I am very glad to get response from both of you! Thanks!

First, I am reading a lot of paper about the molecular X and I found, that some of paper have found the downstream pathway in a single cell, which means the intracellular domain is known. However, our lab wanna to do research in the extracellular domain. Our first attempt is to transfect the overexpressed molecular X to cells and enrich the membrane. Than use the membrane to co-IP or mass spectrum. I will finish the assay and find the way.

Another question, does the dowanstream pathway molecular mean "a ligand" ,either?

Or, does the microglia which activate the molecular X mean "a ligand"??

I think I am confused about the conception of "ligand".....

I hope to get your advises, thank you !!

Miao- without knowing the specific details of which proteins you are working with, I don't think I can make additional suggestions. However, ligand simply means something a protein binds. This can be either a small molecule (hormone, metabolite, drug) or another protein. Do not confuse ligands with substrates. Substrates are also bound, but transiently, such that the protein can transport them or catalytically modify them. In the context of signal transduction cascades, downstream molecules are more likely to be substrates than ligands.

Thanks to Daniel, I think can identify the difference of between substrate and ligand. You said the substrate is transient bound, and I am wondering whether the ligand-bound is forever?

No, nothing is bound forever. And other than biotin-avidin, and rapamycin-FKBP interactions, very few ligand-receptor interactions have binding half-life that last days/weeks/years (perhaps in relativistic terms you might call these interactions "forever", even if not strictly true). The half-life of the receptor-ligand complex is a function of the affinity (low in this case?), ligand concentration, plus additional regulatory mechanisms such as endocytosis, degradation, or cleavage of the receptor-ligand complex.

Whether a downstream signaling target could be a ligand for your membrane protein is hard to judge without more information, but in general, interactions with downstream signal transduction targets are transient (millisecond-to-second time scales).

I get it, Daniel, you are great helpful to me! Thank u and wish u luck in your research life!^_^

I'm a bit confused by the question. If you know the ligand as a low affinity for the receptor then you must know what the ligand is. Are you trying to find novel ligands? If you have candidates you might want to try cross linking reagents or see if there are azido-derivates that will covalently bind to the receptor