BCH 499 – Research II
Number of credit hours: 2
Number of students with me - 5
In this course the students are given small projects and taught how to plan and carry out a given experiment. At the end the students are required to write a report and have an oral exam.
Experiment in the current semester
Extraction and determination of cholesterol from brain of various mammals viz.,Arabian camel (Camelus dromedarius), sheep, goat, rabbit and cow.
Estimation of cholesterol in tissues
- Sulphuric acid
- Glacial acetic acid
- Sodium chloride (0.05 M)
- Calcium chloride (0.36 M)
- Ferric chloride – 2.5% w/v in phosphoric acid
- Phosphoric acid
- Cholesterol standard – 1 mg/ ml. Dissolve 100 mg of cholesterol in chloroform/methanol mixture (2:1) and make the volume to 100 ml.
- Test tubes – approximately 50 numbers
- Pasteur pipette
- Glass cuvettes
- Motor & pestle or blender
Extraction of Cholesterol from the tissues
- Weigh 100 g of tissue.
- Homogenize 100g of tissue with chloroform/methanol (2:1) in waring blender and make the volume to 100 ml or 1.0 g of tissue in chloroform/methanol (2:1) and make the final volume to 20 ml.
- Centrifuge at 3000 rpm to remove the precipitated proteins.
- Remove the supernatant.
- Wash the supernatant with NaCl (5 ml) and centrifuge at 3000 rpm for 15 minutes.
- Separate the lower layer (this contains cholesterol) using Pasteur pipette.
- Wash this lower layer with CaCl2 /methanol (1:1) .
- Centrifuge at 3000 rpm for 10 minutes
- Use the lower layer for cholesterol estimation.
- Note the volume of the final extract.
Estimation of cholesterol
- Take 1.0 ml of the extract in test tube
- Add 3.0 ml of glacial acetic acid
- Add 0.3 ml of ferric chloride
- Add 3.0 ml of concentrated H2SO4
- Mix the tubes slowly and allow it to stand at room temperature for 10 minutes.
- Read the absorbance at 560 nm using glass cuvettes.
Preparation of standard
Stock standard solution – Weigh 100mg of cholesterol and dissolve in chloroform/methanol mixture (2:1) and make the volume to 100 ml. (Concentration is 1mg/ml). This is solution A.
Working standard – Pipitte 10 ml of stock standard (Solution A) and make the volume to 100 ml with chloroform/methanol mixture (2:1). (Concentration is 100µg/ml). This is solution B.
Weight of the tissue - ________g
Volume of the final extract - ________ g
Note – The reagents are strongly acidic and must be handled with extreme care.
Transfer to cuvettes very carefully and do not allow any liquid to drop along the outside of the tubes. Wash any spills immediately with a solution of biocarbonate followed by generous amount of water.
An Introduction to Practical Biochemistry by David T. Plummer, third editionTata McGraw-Hill Publishing Company Limited, New Delhi, 1990.