If your protein is toxic for E. coli it is probable that tagged protein was also toxic. However, notice that to generate antibodies you don't need to express the whole protein, you can try different fragments and probably some of them won't be toxic for overexpression. You only should take care about the fragment election (they should be specific of the protein to ensure the specificity of your antibodies).

Other option is to buy a commercial synthetic peptide of your predicted protein, I have never used them before but some people do and have suitable results.

Hope this help.

Good luck.

A common strategy is to generate a chimera protein containing your inV gene attached to a tag (GFP, HA, HIS, FLAG are quite common but there are others). This way you can pull down your protein of interest using the widely available commercial antibodies for these tag proteins. It might be worth doing both N-terminal and C-terminal versions depending on what you want to do after purification.

If your protein is toxic for E. coli it is probable that tagged protein was also toxic. However, notice that to generate antibodies you don't need to express the whole protein, you can try different fragments and probably some of them won't be toxic for overexpression. You only should take care about the fragment election (they should be specific of the protein to ensure the specificity of your antibodies).

Other option is to buy a commercial synthetic peptide of your predicted protein, I have never used them before but some people do and have suitable results.

Hope this help.

Good luck.

The strategies commented from both Daniel and Carlos are suitable, and deciding from one or the other would depend in the time you want to spend. Generating an antibody takes time and you will have to test that you could use it for your needs.

A common strategy, when you want to avoid this, would be to generate a chimera with a suitable tag or combination of tags. There are many ways, but you shoud think that you may always carry some contaminations over. An approach would be to use different tags in tandem separated by endoprotease cleavage sites. You could overcome the toxicity problem if you could engineer the tags directly in the genome of the enterobacter, if that is possible, and do the purifications directly from them. This system works nice in yeast and had been used to identify the molecular partners in genome-wide analysis.

Hope this helps. Good luck.

If you want to avoid using TAGs, for this may interfere with the native folding and activity of the cell, you will need to develop a thorough protocol to purify your protein in order to isolate enough of it to generate antibodies to your protein (companies and some labs, such as the one I work in, can make antibodies for you for a reasonable price but first we need a good amount of purified protein). However, I think if you can get away using a TAG this will save you a lot of time.

To isolate your protein you need to consider some of its innate characteristics(chemical, physical and biological characteristics) that make it unique and exploit those characteristics. You can use 2D electrophoresis to separate proteins by their mass and isoelectric point. You can use Ion-exchange chromatography to separate proteins based on their polarity. You can separate proteins based on their solubility characteristics (membrane vs. soluble proteins). There are really good protocols already out there used for protein purification. Once you have developed a protocol that will isolate and will give you a good yield and good quality of your protein, generating antibodies is an easier process. Though the initial amount of work is cumbersome, if you stick it out the payout will be worth it. I have antibodies to proteins I often used and I can purify the proteins for biochemical studies in one step via immunopurification, the process is quick and gives me a good yield of protein every time.

Good luck!

Clone the protein coding gene into vectors like Daniel said. pull down using the specific antibody and resigns for eg: His tag with Ni-resin.

Best

Surakasi

all options require the proteins to be expressed and if it is toxic to the cell this will be a veeery difficult procedure till it works... but maybe you can purify higher amounts from the liquid media after the cells died?

If you want to have antibodies you should try synthesizing a peptide of your protein and raise antibodies against that - fastest way (but not very likely to work well)

Maybe an in vitro protein expression system could work for you?

Can you express your protein in an organism that is not affected/killed by it? I think Carlos's suggestion is probably the best one so far.

try cloning your gene into a baculovirus expression system with an appropriate N- or C-terminal TAG. This will allow high accumulation of your protein in the extracellular environment while limiting toxicity. It also allows high purification using specific antibodies against your "TAG". Alternatively, you could clone a TPA (or alternative) secretion/exporter peptide leader onto the gene for your protein of interest which would also allow for the accumulation of your protein in the extracellular environment. However, the baculovirus expression system is very optimized and easy to generate high levels of proteins quickly. I attach information for you below.

Does it has a known protein partner that you could use as a bait? Or does it bind to DNA? Last, but not least: have you considered in vivo tagging? Good luck!

If your protein is toxic, try an expression system that is tightly controlled, e.g. the arabinose-inducible strains BL21 AI, available from Invitrogen (Gibco). It works better than BL21(pLysS), which has a significant leakage expression, even in the absence of IPTG

You can produce polyclonal antibodies raised against peptides and not the protein. The design of the peptides is very important but most of companies propose this service. Informations about the 3D structure of the protein (or a similar one) helps for the design of the peptides. The best and more secure is to immunize a host against 2 peptides.

thank you everyone. i will try to follow what Carlos suggested. i think that would be pretty much feasible in my system. thank you for the other suggestions as well.

cheers!!!