## 4 Discussion
### Kdo\({}_{2}\)-Lipid A Extraction and Deacylation  
As discussed in the protocol the cell dry-down and lipid extraction can be done from fresh or frozen cells. Cell pellets can be frozen at \(-80\)\({}^{\circ}\)C for up to one month prior to use due to the stability of the Kdo\({}_{2}\)-Lipid A. It is emphasized in the protocol to fully resuspend the cells when drying them down in each of the solvents before centrifugation. This is intended to make sure that all the cells are exposed to the solvent on their cell surface, since the drier the cells, the more successful the extraction will be. If care is not taken during the dry-down step, it will yield a clumpy beige/grey cell solid that is difficult to work with. Lipid extracted from these cells is gummy and requires a lot more wash steps than usual which inevitably reduces overall yield. Once successfully dried, the cells can be stored in a Falcon tube or capped vial at 4 \({}^{\circ}\)C for about a month before the extraction. The storage environment must be a dry because exposure of the dried pellet to moisture will cause molding of the cells. Pulverizing the dried down cells is a necessary step prior to the lipid extraction, however, pulverizing the cells will also generate more surface area to ensure efficient resuspension in the extraction solution. Cell resuspension is done initially by spatula, then by aggressive vortex, and finally by circulation on a nutator. The extracted solution should appear clear and yellow with no floating particulate matter, and if necessary, a second filtration step can be performed to remove any visible solids as they will also negatively impact this process. Removal of petroleum ether and chloroform by way of rotary evaporator is a critical step that if overdone will breakdown the lipid in solution before it can be successfully precipitated. To avoid this, high vacuum conditions, with rapid rotation in a room temperature water-bath for \(-10\)-\(15\) min will remove the more volatile solvents leaving a yellow crude extract that is deeper in color, but not dark. Be sure to give the solution sufficient time to allow full precipitation of the lipid from solution, a minimum \(45\) min is recommended but to obtain a much higher yield the mixture should stand overnight at room temperature before centrifugation. As mentioned previously, a large volume of the pellet will dissipate as the washes continue; this is to be expected as the extracted pellet contains components of the lipid bilayer as well as other membrane bound lipids which are undesirable in the final product. If there is any undissolved lipid in the \(1\%\) TEA, it will not lyophilize properly and therefore will not deacylate to completion, which is why it is important to add a little solution at a time until the lipid dissolves fully. The KLA solid will be a fluffy white solid that appear cotton-like in texture; these characteristics are a good visual cue of purity as well as characterization via mass spec. Due to the highly charged nature of the compound, it will often be detected as the doubly or triply charged species, and have a correspondingly smaller observed \(m/z\). The compound can also be characterized via NMR if it is to remain in it is fully acylated state. Kdo\({}_{2}\)-Lipid A is not fully water soluble and will require the use of detergent for experimental purposes. Chaps and Triton X-100 are two commercially available detergents that have been shown to work well with Hepl and similar proteins that will solubilize the Kdo\({}_{2}\)-Lipid A [34]. Experimentally, it has been shown that selective deacylation generates a hydrophilic lipid A moiety that does not differ greatly from the fully acylated version in it is binding and kinetic interactions with the protein [25]. The deacylation reaction is straightforward and dependable, as hydrazine is a potent reactant if stored properly and extracted from the reagent bottle under nitrogen while not introducing any water contamination.