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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

ISOLATION AND STORAGE OF RAT SERUM CONJUGASE FOR TISSUE FOLATE ANALYSIS

Isolation and Storage of Rat Serum Conjugase for Tissue Folate Analysis
 
Overview
This protocol describes how to prepare and store rat serum conjugase for use in folate analysis. Rat serum conjugase removes the poly-glutamate residues from the folic acid found in tissue extracts. This renders the folate available for utilization by Lactobacillus casei in the microbiological assay of tissue folate content.
 
Procedure
1. Collect blood from normal rats by cardiac puncture with a syringe and 16-gauge needle without anticoagulant and place it into a 15 ml conical tube (see Hint #1).

2. Incubate the tubes on ice for 10 min to allow the blood to clot.

3. Remove as much of the clot from the tube as possible and centrifuge the tubes at 5,000 X g (i.e. in a clinical centrifuge at full speed) for 10 min at room temperature.

4. Transfer the serum to 50 ml conical tubes and add 5 g of Activated Charcoal for every 100 ml of serum collected.

5. With occasional mixing, incubate the serum/Activated Charcoal solution for 1 hr on ice.

6. Centrifuge the tubes at 5,000 X g for 10 min at room temperature.

7. Recover the supernatant and filter it though a 0.22 μm filter.

8. Store the rat serum conjugase in 250 μl aliquots at -20°C.

Solutions
This bioProtocol does not use any solutions
 
BioReagents and Chemicals
Activated Charcoal
 
Protocol Hints
1. Approximately 12 ml of blood can be harvested from a 250 g rat. Hemolysis is to be avoided at all costs. Thus, do not pull too hard on the syringe while withdrawing the blood. Blood from multiple rats can be collected at one time and processed in batch format.

 
Citation and/or Web Resources
1. Wilson SD, Horne DW. Use of glycerol-cryoprotected Lactobacillus casei for microbiological assay of folic acid. Clin Chem 1982; 28:1198-200.