Principles of Biochemistry (2+1)

Estimation

Into each of two test tubes, pipette the following

Reagent (ml)	Test	Blank	Test	Blank
Water Lipase substrate Trizma buffer Extract	2.5 10   1.0 1.0	2.5 10   1.0 --	2.5 3.0   1.0 1.0	2.5 3.0   1.0 --

 

• Cap with clean rubber stopper and shake vigorously for 5 sec.

• Place both the tubes in a constant temperature water bath or incubator at 37ºC. 
•  Note the time.
• Incubate for 3 hrs. 
• Immediately after starting the incubation, pipette 1.0 ml extract into a 50 ml Erlenmeyer flask.
• Mark the flask as BLANK and store in refrigerator (2-6ºC).

• After the incubation period of 3  hrs.

• a) Pour the contents of BLANK tube into the BLANK flask

• b) Pour the contents of TEST tube into a clean 50 ml Erlenmeyer flask, cap with clean rubber stopper and shake vigorously for 5 sec.

• Into each of the two tubes (BLANK and TEST) pipette 30 ml 95% ethanol, shake to rinse, and pour into their respective flask.

• Add to each flask 6 drops  of thymolphthalein indicator solution.

• Fill up a burette with 0.05 N NaOH solutions. Note initial burette reading.

• Titrate each flask to a slight but definite blue (not dark blue) colour.