Whether a membrane protein produced in a cell-free system will at all fold to the native conformation and/or can be reconstituted into nanodisks in native form depends very much on the particular protein. (By the way, urea is a denaturant, not a detergent). Whether the protein can be renatured with a reasonable effort, whether in the presence of detergents or nanodiscs, has to be tested experimentally, unless a corresponding procedure for this particular protein has already be published.

Depending on the way the protein is embedded in the membrane, you might be able to express individual domains of the protein that are able to fold and do not contain the transmembrane part, or you might be able to raise antibodies crossreacting with the native form of the antigen by immunizing with peptides representing exposed loops of the antigen.

If you inject the protein in nanodisks, you will probably also get a lot of antibodies recognizing the protein component of the nanodisc. Another problem with immunizing with membrane proteins is that your antiserum might contain a large fraction of antibodies against the hydrophobic, normally membrane embedded part, which tend to stick unspecifically to hydrophobic surface.

Whether a membrane protein produced in a cell-free system will at all fold to the native conformation and/or can be reconstituted into nanodisks in native form depends very much on the particular protein. (By the way, urea is a denaturant, not a detergent). Whether the protein can be renatured with a reasonable effort, whether in the presence of detergents or nanodiscs, has to be tested experimentally, unless a corresponding procedure for this particular protein has already be published.

Depending on the way the protein is embedded in the membrane, you might be able to express individual domains of the protein that are able to fold and do not contain the transmembrane part, or you might be able to raise antibodies crossreacting with the native form of the antigen by immunizing with peptides representing exposed loops of the antigen.

If you inject the protein in nanodisks, you will probably also get a lot of antibodies recognizing the protein component of the nanodisc. Another problem with immunizing with membrane proteins is that your antiserum might contain a large fraction of antibodies against the hydrophobic, normally membrane embedded part, which tend to stick unspecifically to hydrophobic surface.

Definitely you will have aggregation if you do not use nanodiscs in the system.

However to raise antibodies you don't really care about preserving conformation so you may be "safe" just recovering the protein aggregates.. but it depends what are your specific goals (what else will u do with the protein?)

consider also that in order to raise antibodies you may use just small peptides.. that are much easier to express and purify rather then the full length protein and can be easily expressed in a normal expression system (bacteria/yeast) which are much cheaper then cell-free systems.

In any case, how do you plan to purify the protein? You must do so to remove all the contaminants and, in this case, you normally have the protein in detergent micelles!

Dear Vicken,

I agree with Annemarie, you might get a lot of MSP-specific antibodies from the nanodisc scaffold itself. However, I don't recall many reports from immunizations of animals with nanodiscs so it's worth a try.

Definitely I would not recommend using unfolded/refolded or detergent-resolublized material, unless you specifically need an antibody for unfolded protein or a linear epitope.

If reconstitution of your protein into nanodiscs or nanodisc-based immunization poses a problem, you might consider reconstituting your membrane protein directly into liposomes which are a standard medium people use for immunization of animals with integral membrane proteins. If I recall correctly there are protocols for reconstitution of cell-free express protein directly into liposomes.

Also, if the linear epitope is to be recognized, immunization with a peptide, like Paola suggested is a viable alternative.

Finally, if you see an efficient reconstitution of your membrane protein into nanodiscs, you might consider immunization-free strategies to obtain antibodies. These are based predominantly on phage or yeast display methodologies which work well with membrane proteins in nanodiscs.

Best,

Pawel

Thank you for your suggestions,

Like you have suggested i am just going to go with synthesized peptides.

It seems the best option.

another option -depending on the protein you are working with- is to express soluble parts of it fused to purification tags (such as maltose binding protein or GST) in E. coil. Next, purify the fusion protein and use it for immunizations. of course, the polyclonal antibody will recognize both the tag and your protein, but this in most cases does not any problem at all. the advantage over peptides is that the part of the antigen that corresponds to your protein can be much bigger than the 17 amino acids commonly used for peptide-based immunizations. for immunizations, the peptides are usually coupled to bigger protein (e.g. BSA) so you will have unspecific antibodies in both cases.

all the best

Jürgen

You first need to consider what you need the antibody for. Rule of thumb: If you want to use the antibody with native protein (say, neutralisation or immunoprecipitation), use native antigen for immunisation. If you want your antibody to react with denatured protein (say, for western blotting), use denatured antigen to make the antibody. In either case you may be lucky and get antibodies that work in both types of situation, but that is not the rule (especially if you want monoclonals).

Nanobodies are wonderful for studying membrane proteins because of their very precisely defined composition. For cell-free translation, however, I'd probably use preparations of rough ER, that contain docking protein (SRP-receptor).