## 3 Results and Discussion
### Purification of the Crude Protein Extract  
Partial purification of the crude extract using ammonium sulfate precipitation removed proteins that did not precipitate at 40-60% salt saturation. This also concentrated the dilute crude extract, shown by more intense protein bands (Figure 1, lane c), through the dissolution of the precipitated proteins in a smaller buffer volume [4]. Dialysis, on the other hand, removed most of the ammonium sulfate present in the partially purified sample which could affect the results of the succeeding protein characterization procedures. Lastly, gel filtration chromatography (Figure 1, lane d) as a purification procedure effectively separated substances with MW range between 10 kDa to 100 kDa which refers to the effective separative capacity of the resin used.  
Comparison of the protein profiles in Figure 1 (lanes b, c and d) showed that the purification scheme was effective. Protein band at 10 kDa and other faint protein bands at 25-37 kDa area of the crude profile were not observed in the purified profile. Moreover, the protein band at 15 kDa became more distinct after purification.  
Densitometric analysis allowed estimation of the molecular weights and calculation of the percent band volume of each protein present in the SDS-PAGE profiles of the crude, partially purified, and purified protein extracts. This showed that the major proteins have MW of 15 kDa, since this protein band had the highest percent band volume ranging from 26% to 34% per sample lane.  
Data from Table 1 shows that there was a significant decrease in protein concentration after every purification procedure. This means that non-major proteins were subsequently removed from the crude and partially purified extract leaving only the major proteins in the purified extract. Although the results of the colorimetric Bradford assay in Table 1 showed a decrease in concentration after every purification step, the SDS-PAGE profile of partially purified (lane c) and purified extract (lane d) in Figure 1 had more intense bands compared to the crude extract (lane b). This is due to loading of preconcentrated partially purified and purified extract into the gel system. If preconcentration of the said extract were not done, their dilute concentration might result to very faint protein bands. Meanwhile, the crude extract was not pre-concentrated prior to SDS-PAGE since it already gave a workable protein profile band with the concentration as it is.  
Expounding on the final purification procedure, Gel Filtration Chromatography (GFC), Figure 2 shows that there was only one observable curve which peaked at fractions 20 and 21. An elution profile showing a curve or band rather than being narrowly peaked suggests that the purified protein was distributed in the fractions found under the curve rather than on a single fraction. SDS-PAGE result of these fractions showed the same profile with protein bands becoming more intense from the 19 th up to the 23 rd fractions (refer to Supplementary Materials). The fractions outside the curve have no visible protein bands.  
Reconsidering Figure 1, the distinct protein band found at 10 kDa of the crude and partially purified protein extracts did not appear at the purified extract. Moreover, the said protein band did not also appear at any GFC fraction even though there were already 60 fractions collected. This protein band did not elute out of the column as the resin only effectively separated proteins with MW between 10 kDa and 100 kDa. Proteins with molecular weight smaller than the given range would be trapped and totally included in the pores of the gel beads with the possibility of not being eluted out by merely using the same elution buffer.