Protein Content

Safety measures

• The work should be conducted using the FOSS Kjeltec 8100 in the analytical laboratory at Tel-Hai
• The work should be scheduled with Shai - the manager of the analytical lab (050-5328156)
• The work should be conducted in chemical hood
• When dealing with hot equipment and sulfuric acid - wear thick gloves

Equipment

• Analytical balance
• Kjeldahl tubes (at the number of samples)
• Gloves for high temperatures

Materials

• NaOH (0.1M)
• HCl (0.1M)
• Sulfuric acid
• Hydrogen peroxide (30%)
• Standard solution (provided by Shai)
• Receptacle solution (provided by Shai)

Protocol

1. Weight approx. 0.2 gr of material (dry) and place in Kjeldahl tubes.
2. Add 4 ml of sulfuric acids using dispenser and place in the hood for 12 hr.
3. Place the tubes in hot bath, 275 degrees for half an hour.
4. Add Hydrogen peroxide (30%) and wait for 10 min until the solution will be cleared (if it is not cleared - repeat this stage)
5. Remove the tubes from heating and wait until cooled to room temperature
6. Complete the volume in the tubes to 100 ml with distilled water

working with FOSS Kjeltec

1. Turn on the Kjeltec machine
2. Place empty tube in the distillation unit and close the door, put empty erlenmeyer bottle at the distillate exit point
3. Run program 10 to heat the machine
4. Run a standard solution (provided by Shai) - if wasn't run for a long time
5. Put 25 ml receptacle solution (provided by Shai) in the erlenmeyer and place the bottle at the distillate exit point
6. Place the tube with the sample in the distillation unit and close the door. Run program 3. At the end of the process the color of the receptacle solution will be green
7. The receptacle solution should be titrated using HCl (0.1M) until the receptacle solution color changes to grey
8. At the end of usage or between biological replicates, an empty tube should be running in program 6
9. Calculation of amount of protein should be done using

Calculation of % Nitrogen

• MW of nitrogen (g/mol) – 14.0067
• Titrant normality (0.1N) – 0.1
• and using factor 6.25 [[[1]]]