Wetlab Protocol

1. SDS PAGE
   

   SDS-PAGE analysis will running using 8-16 % Tris-glycine acrylamide gradient gels (Novex, San Diego, CA). The culture will prepared by mixing 25 µl of 2x loading dye with 25 µl of each cell suspension. Than the sample enter to the gel and also protein ladder. The solution will be heated at 100oC for 5 min. To each lane of gel, 10 µl of dye-sample mixture will be loaded and the electrophoresis perfomed in SDS-Glycine buffer at 130V constant until dye reached the bottom of the gel (Sambrook et al., 2003).



2. Ethylene Glycol Assay using Chromatic Acid
   1. Ethylene Glycol Standard Solution Procedure
      1. Ethylene Glycol concentration ≈ 0.5 M (31035 ppm) -> Dissolve 279 µL ethylene glycol in 10 mL deion water.
      2. Ethylene Glycol concentration ≈ 0.05 M (3103.5 ppm) -> Dissolve 300 µL ethylene glycol 0.5 M, then dilute until 3000 µL.
      3. Ethylene Glycol concentration ≈ 0.005 M (310.35 ppm) -> Dissolve 300 µL ethylene glycol 0.05 M, then dilute until 3000 µL.
      4. Ethylene Glycol concentration ≈ 0.0005 M (31.035 ppm) -> Dissolve 300 µL ethylene glycol 0.005 M, then dilute until 3000 µL.
      5. Ethylene Glycol concentration ≈ 0.00005 M (3.1035 ppm) -> Dissolve 300 µL ethylene glycol 0.0005 M, then dilute until 3000 µL.
   2. Chromic Acid Standard Solution Procedure
      1. Chromic acid ≈ 0.6 M (177000 ppm) -> Dissolve 1.77 gr K2CrO7 (Mr 294.18) with sulphate acid until the volume 10 mL.
      2. Do dilution of chromic acid same as ethylene glycol standard solution procedure.
   3. Ethylene Glycol Concentration Measurement through Spectrofotometri UV-Vis
      4 Cr2O72- + 3 C2H6O2 + 32 H+ -> 8 Cr3+ + 3 C2H2O4 + 22 H2O
      1. Ethylene glycol 300 ppm -> Pipette 29 µL ethylene glycol solution 31.035 ppm then dilute it until 3 mL.
      2. Chromic acid 3.000 ppm -> Pipette 8.5 µL chromic acid solution 177.000 ppm then dilute it until 5 mL.
      3. Sulphate acid blank solution -> Enter 1 mL sulphate acid into cuvette, then search maximum wavelength for this solution.
      4. Chromic acid blank solution -> Enter 1 mL chromic acid 300 pm into cuvette, then search maximum wavelength for this solution.
      5. Add 600 µL chromic acid 300 ppm and 400 µL ethylene glycol, then measure the absorbance of this solution.
         The difference absorbance values between chromic acid were added with sulfuric acid and ethylene glycol can be used as a parameter to calculate the concentration of ethylene glycol in the sample.
   4. Qualitative and Quantitative Ethylene Glycol Measurement
      Qualitative Procedure:
      1. Add 0.25 mL sampel into microtube
      2. Add 0.5 mL Acetone and 0.1 mL Chromic acid (H2CrO4) into microtube
      3. Mix the mixture
      4. Incubate the mixture in waterbath at 65 - 700C for 10 minutes
         Positive result will show the color changes from orange to green
      Quantitative Procedure:
      1. Add 0.25 mL sampel into microtube
      2. Add 0.5 mL Acetone and 0.1 mL Chromic acid (H2CrO4) into microtube
      3. Mix the mixture
      4. Incubate the mixture in waterbath at 65 - 700C for 10 minutes
      5. Check the absorbance at A446 nm