## 3 Results
### Batch Adsorption of the Active Protein from Cell-Free Filtrate Dialysate to Ion-Exchange Resins  
The strain NH CFCSAS precipitate dialysate was tested for binding to ion-exchange resins at 10 different pH values, as described in the materials and methods. These experiments indicated that the bactericidal protein bound strongly to a cation-exchange resin at an acidic pH, but relatively poorly to an anion-exchange resin at all pH values (Figure 5). This information was subsequently used to partially purify the bactericidal protein via cation-exchange chromatography.  
Figure 4: Gel filtration chromatography of the strain NH CFCSAS precipitate dialysate. **(A)** Size standards: 1 = chicken ovalbumin (44 kDa); 2 = horse myoglobin (17 kDa); 3 = vitamin B\({}_{12}\) (1.35 kDa). \(n=3\). **(B)** Bactericidal fraction cumulative volumes and corresponding mean and SD for the molecular weight, \(n=2\).  
Partial Purification of the Bactericidal Protein via Cation-Exchange Chromatography and Isoelectric Focusing  
The strain NH CFCSAS precipitate dialysate, equilibrated in 50 mM acetate buffer at pH 5.0, was loaded onto an equilibrated 30 mL SP-Sepharose column. Following washing of nonspecific material off of the column, bound proteins were eluted from the column with an isocratic 0-500 mM NaCl gradient in 50 mM acetate at pH 5.0. Fractions from the column elution were quantified by BCA protein assay. Protein peak fractions (Figure 6A) were pooled and concentrated by ultrafiltration (with a cutoff of 10 kDa). Each protein peak was then tested for bactericidal activity against _C. jejuni_ (Figure 6B). Inactive and active peaks were then compared by SDS-PAGE to see if any protein bands correlated with the bactericidal activity (Figure 6C). An 11 kDa band on SDS-PAGE appeared to be enriched in the active peak and absent or very diminished in protein peaks that were found to be inactive in the bactericidal activity assay. Separation of active peak proteins by isoelectric focusing (Figures 7 and 8) and bactericidal activity assay of focused fractions confirmed the association between the presence of the 11 kDa band and bactericidal activity against both _C. jejuni_ and _L. monocytogenes_.  
Figure 5: Batch adsorption of anti-_Campylobacter jejuni_ bactericidal activity to ion-exchange resins at various pH values. Activity units (AU) = reciprocal of the endpoint dilution (greatest dilution inhibitory to _C. jejuni_) per mL of fraction tested. Bars and brackets represent the mean and standard error of three independent experiments, respectively.  
Figure 6: (**A**) Protein elution from the cation-exchange column; (**B**) Bioassay on peak protein fractions (63, 71, 80, 89, and 99) from elution of cation exchange column. Numbers in cells of rows A and B are absorbance at 600 nm of _C. jejuni_ cells, and proportional to _C. jejuni_ growth. Inhibition of growth by active peak 99 is indicated by yellow highlighting. PC: positive control well lacking _Camyylobacter jejuni_; (**C**) SDS-PAGE with 10 \(\upmu\)g/lane of protein peaks from the cation exchange column. Arrow points to enriched protein band in the active peak, fraction 99.  
Figure 7: Isoelectric focusing of protein peak active against C. _jejuni_ from cation exchange column, trial 1. (**A**) pH of fractions, trial 1; (**B**) bioassay, trial 1; (**C**) SDS–PAGE trial 1: dialysate (lane 1), active peak from cation-exchange column and isoelectric focusing fractions 8, 5, 6, and 7, with each lane loaded with 10 micrograms protein.  
Figure 8: Isoelectric focusing of protein peak active against _L. monocytogenes_ from cation-exchange column, trial 2. (**A**) pH of fractions, trial 2; (**B**) bioassay trial 2; (**C**) SDS–PAGE trial 2: active peak from cation-exchange column and IEF fractions 5, 6, 7, 2, 3, 4, and 9, with each lane loaded with 10 micrograms protein.