A bit of a long shot, but you could label the total cell surface expression with a specific antibody, wait for a few different time points and lyse the cells. An ultracentrifugation protocol for separating the cytoplasm and the membrane will separate out the internalised cytoplamic preparation. You could then use this cytoplasmic fraction in an ELISA? Only internalised protein would have the antibody bound and show up in the ELISA. Although I am not sure that the antibody would remain attached to the internalised protein during the lysis.

Like John Mentioned, if you expect that your antibody is on the surface and after sometime it is internalized, you could probably label your membrane bound to antibody with biotin, so if your antibody has any lysine residue, biotin will bind to that. Lyse the treated HEK cells with the lysis buffer, take the lysate and incubate with avidin or streptavidin beads. The bound forms should be your antibody bound to membrane and then when you centrifuge that conjugated lysate, whatever comes out is your cytoplasmic fraction. so if you do ELISA with these two fractions, may be you can get an idea how much was bound and how much has been internalized. I am not very sure of the specificity though as biotin could label any peptide or protein and specificity is checked only with peptide specific antibody.

Maybe digesting surface bound antibody with a protease like trypsin while inhibiting internalization by cooling to 4degC is helpful. Of course, you'll need to run time series.

To obtain a non-internalized, membrane bound, control sample, incubate your cells with AB at 4degC to inhibit vesicular transport. This could be a good starting point for the rest of your experiment, by running the time series eg in an mis-used thermocycler.

Good luck and have fun!

Wo.

Hello, Anette,

In this publication (http://jpet.aspetjournals.org/content/325/2/544.full.pdf+html) you can find an ELISA based assay for receptor internalization. I assume you can adjust it for your needs.

Good luck.

Below link provides the information about internalization of antibody by ELISA.

http://www.unc.edu/~damania/K1-Endo.pdf

Hi, Can..since Anette wants to look at internalization fraction and generally ABs are receptor mediated endocytosed and hence if say the purpose is to capture that fraction by whatever means of making cells vulnerable to internalization, without lysing and without imaging how can ELISA detect it. I mean the Intracytoplasmic fraction ratio can be detected only when the membrane is ruptured or fixed and stained and visualized. Please correct me if I am missing something here.

Generally when we have a peptide specific AB we dont bother about non-specific labeling as the detection will be based on your primary AB which is specific but here the protein itself is an AB so:

label the AB, allow it to bind to your HEK cells which are seeded on a plate, say a 96 well or whatever is the requirement.

The internalization can be initiated with whatever treatment and then cells could be frozen at that point by lysing and fractions can be pulled down by beads.

another thing could be that ABs can be radiolabeled and the cells can be allowed to interact with the AB. Then they will be lysed and spun, the supernatant can be quantified for the radioactivity (liquid scintillation) which would reflect the labeled AB in cytoplasm Vs an unlabeled control.

Sreedhar's paper ref is a good one, useful.

Ya Can ..answer seems logical one, well if one wants to really confirm that when you get a decrement on the outside, do you actually see and increment inside, its not about localization but correlation. But on the fist go your suggestion is quite doable...:)

Thanks for all advice and suggestions.

I tried to incubate my cells with biotinylated antibody on ice or at 37 degrees. I used an antibody which was labeled with a cleavable biotin. Thereafter I washed and stripped the cells (=cleaved off the biotin from the antibodies still bound to the surface). I thereafter lyzed the cells and tried to detect internalized antibodies using ELISA. I can however not detect any antibodies after lysis. I don't know if it is because no antibodies were internalized or if the biotin is cleaved off during lyzis or if the biotin is cleaved off from the internalized antibodies as well. When I just lyze the cells without stripping them I can detect biotinylated antibodies so I don't think that the lysis as such causes cleavage of the biotin.

I have also tried to do as many of you suggest, comapre the signal from antibody binding on the surface of the cell when incubating with the antiboy on ice or at 37 degrees (I have not yet tried to do a thoughal timeline) and can see a lower signal after incubation at 37 degrees. But can I be sure that this is caused by internalization and not because the biotin is cleaved of at 37 degrees?

I don't need to know the localisation of the antiboy after internalization but I want to know that it is internalized. As I see it comparing the binding on whole cells is the easies way to measure, but it does not confirm that the antibody is internalized.

Thanks Can

Not nonsense at all.

I apply my cell lyzate on streptavidin coated plates, so that problem I had already thought a bit of. I am going to label my antibodies with uncleavable biotin aswell, thinking that I then don't have to consider lossing the biotin.

The degradation of antibody inside the cell is another matter. Because I have no idea of how stable the antibodies are inside the cells or what pathway they take once inside, it would be better to use an assay ith whole cells and just measure the surface binding.

I am not familiar with working with endocytos inhibitors. Do I just supplement the media with them? In what concentration?

Thanks again for the advice.

What i use for my experiments is an esterified form of Biotin EZ linked S-S bition which has high affinity, what you have to look for is that do you want a cleavable biotin tag or not. you need to avoid certain reagents which has tris in it later when you are washing, as some reagents would cleave biotin off and when you actually allow the lysate to be incubated with a resin, the biotin tag would have already gone off. Hence you wont detect anything.

next thing is since you are fishing a specific thing in your whole cell, your conc of lysate matters. Lysate has to be quantified before adding to any bead for better output, and even how much biotin you take initially to label as it will matter when you want to recover the tagged fraction.

Yes knowing your Ab turnover is important if you are not doing a time course labeling. endocytosis is generally a fast process and hence atleast within minutes, the cells are either blocked or the time point is frozen by some treatment where only internalization has happened and no other signaling is going on. Its difficult but can be done as Can mentioned with blockers.

I would suggest an I-125 labeling treatment of your Ab which would give you a lot of time to explore internalization of your Ab with the same labeled lot of Ab. I am not familiar with HEK cells, but when I studied internalization of an Ab specific for human Raji B-cells which have Fc receptors, I would incubate, treat the cells with trypsin to clip any Ab attached on the outside surface of the cells, wash and lyse the cells. Then you have the leisure of column chromatography seperation by molecular weight of the fragments of I-125 labeled antibody. This would give you a sense of the fragments and specific antibodies to Fc, Fab, and Fab'2 fragments' binding to the I-125 labeled proteins would verify the fragments present. If done on a timed basis this would give you a good idea of internalized Ab metabolism if that is your interest..

I reccomend using chloroquine in your assays to inhibit lysosomal degradation.

But interpreting antibody endocytosis is a bit tricky because some cells can internalize antibodies bound to surface antigens through a process called capping.

Steingrimur,

in that case (capping): would it make sense to use F(ab)' or ScFv (single-chain ABs)?

Hi Wolfgang. Monovalent binders do not induce capping, as far as I know.

some questions and suggestions:

1. what is the specificity of your Ab?

2. try to fractionate your cells in different compartments: membrane, cyto, nucleus, then detect your Ab and the purity each fraction by wb. do not forget to test the "washing" fraction

3. to my knowledge, the best "inhibitor" of internalization is cold. using specific inhibitos adds more confusion than solution:)

The anitbody is specific for a receptor, which is proposed to be internalized upon binding..

Sounds like a good idea to fractionate the cells and compare.

Although the internalisation is not Fc dependent, I would go for the native form of Ab and not the one with biotin! You never know how this chemical procedure could affect the intrenalisation pathway. Then, you could dectect your Ab by doing a blot just with your secondary Ab. For the purity of each fraction, there are several specific Abs tfor each fraction that you could use!

Good luck!

Can you use the natural ligand for the receptor? That would make the results a lot "cleaner". Does the receptor have an NPXY sequence?

I can not use the natural ligand for the receptor unfortunately. I am right now trying to conduct the internalization assay by inducing internalization with temperature and inhibiting it by cold. I do however not have any success. I can not see any internalization at all.

I am though not totaly sure of that my antibody is internalized so I would like to have a positive control as well, but do not. Any suggestions?

How impotant do you think that it is to have protease inhibitors in the lyzis buffer? Right now I am using a coctail from Sigma.

I have tied to compare the signal from antibodies bound to the outside of the cells during a time scale, hoping to see a reduction in signal with time indicating that the antibody is internalized, but I can not see any clear difference between my antibody and the negative controll (one I know is not internalized).

Any ideas on how to improve that experiment?

Thats too bad that you cant use the natural ligand.

Since you are stuck with antibodies, I would try either antibodies to transferrin or LDL receptors as positive controls. Those are endocytosing receptors and should be on the cells in sufficient quantity .

If you are looking for internalized Ab, you will have to trypsinize and wash the cells to get rid of any surface bound Ab. Internal Ab will be protected against trypsin and it is important to have inhibitors in your lysis buffer.

You can add chloroquine to the endocytosis assay to help keep internalized Ab intact by inhibiting lysosomal proteinases.

Bear in mind though, if the receptor numbers on your cells are low it will be very difficult to demonstrate endocytosis no matter what technique you use.

Why dont you make a stable overexpression of your receptor? Maybe you can boost the phenomenon!