Analysis with the Help of Protein Gels (SDS- Gels)

1. The Appearance of catalase in the Culture Medium

Picture 1. Catalase expression during Bacillus subtilis growth. Excess culture samples were taken from the Bacillus subtilis at different points during its growth phase. The number on the individual spores give the time the probe was taken (in hours) after the cultural inoculation.  Observe the occurrence of the catalase as time goes by (red stripped box). S: (Standard): Fermentas prestained protein marker (#Sm0671), applied quantity: 5µl; M9M: Bacillus subtilis culture, Medium: Minimal medium M9, given amino acids: methionine; LBM: Bacillus subtilis culture, Medium: full medium LB, given amino acids: methionine; LBN: Bacillus subtilis culture, Medium: full medium LB, given amino acids: norleucine. 32 µl of Excess culture samples were placed on the gel (+ 8 µl 5 x SDS diluent buffer).

In the three example cultures of M9 with methionine, LB with methionine und LB with norleucine an occurrence of the catalase can be clearly seen between 7 und 20 hours after the culture inoculation (see picture 1, red stripped box).

Picture 2. Catalase expression after 21 hours. Bacillus subtilis culture residues were applied after the end of the expression time. S (Standard): fermentas prestained protein marker (#Sm0671), applied quantity: 5µl; LB: full medium LB; M9: minimal medium M9; M: methionine; E: ethionine; N: norleucine. 20 µl of each culture were applied to the gel (+ 5 µl 5 x SDS diluent buffer).

All excess culture samples were applied to a gel for comparison

after the end of the catalase expression. Catalase was found in all samples (see picture 2, red stripped box). Comparison of the total protein concentration of the six samples shows that all excess cultural samples held similar amounts of protein. The conclusion can be made that that band intensities of the catalase in the individual probes can be directly compared to one another. Equal amounts of catalase are also in the remains of LBN, M9M, M9E and M9N and also in the remains of LBM and LBE. Comparing the two groups to each other shows that LBM and LBE held approximately 3 times as much catalase as LBN, M9M, M9E and M9N.
For closer inspection, the catalase was removed from the culture residue.

2. Filtration of the Catalase

Picture 3. Two tiered ammonium sulfate precipitation of the catalase. S (Standard): fermentas prestained protein marker (#Sm0671), application amount: 5µl; M9E: Bacillus subtilis culture, Medium: minimal medium M9, defined amino acids: ethionine; LBE: Bacillus subtilis culture, Medium: full medium LB, defined amino acids: ethionine; Ü1: Residue from culture; P1: precipitate of Ammonium sulfate allowance up to 50 %; P2: precipitate of Ammonium sulfate allowance up to 80 %; D1: dialysate from precipitate P1; D2: dialysate from precipitate P2. 20 µl of each culture was applied to the gel (+ 5µl 5 x SDS diluent buffer).

In an initial step, the residues (see picture 3, Ü1) were placed in a concentration of 50%

ammonium sulfate. The precipitates (P1, see picture 3) were separated through filtration. A subsequent ammonium sulfate was added to the resulting residue for a concentration of 80%. The precipitate (P2, see picture 3) was separated through filtration. P1 and P2 were subsequently re-suspended in 10 ml Tris diluent buffer and dialyzed a gainst Tris diluent buffer over night at 4°C (Dialysate D1 und D2, see Picture 3).
Picture 3 shows the exemplary filtration of the culture residues M9E and LBE. Because of the first step of ammonium sulfate precipitation (precipitate P1), the contaminated proteins could be removed from the sample. Especially that in residue (Ü1) more commonly found protein with approximately 37 kDa could almost be completely removed (see black arrows in picture 3) The catalase could be removed from the culture residue by a subsequent addition of ammonium sulfate (see red arrows in picture 3). The subsequent dialyzation brought the catalase into solution (see dialyzate D2 in picture 3).
If the relationship of catalase to total protein in Ü1 and D2 is compared, it is clear that the catalase was highly concentrated by the two tiered ammonium sulfate precipitate vis-à-vis the other proteins in the culture residue.

The dialyzate D2 was drafted for further analyses. Due to time constraints, only the mass spectrometric analysis of the LBE sample could be conducted before the completion of this report.