Experimental extraction of calmodulin from chicken gizzard

Summary

Calmodulin is found in all mammalian tissues and also in other eukaryotic organisms. It is one of the most ubiquitous and highly conserved proteins known. This is because the gizzard is an abundant and inexpensive source of smooth muscle without much vascularization, fat, or connective tissue. And since smooth muscle is an excellent source of calmodulin, and since mammalian and avian calmodulin are identical, using chicken calmodulin would not jeopardize its specificity. This experiment was derived from the Experimental Guide to Protein Purification and Identification, by Zhu Houzhu.

Operation method

Experimental extraction of calmodulin from chicken gizzard

Materials and Instruments

Gizzard 2-mercaptoethanol Buffer H(10X)
Polypropylene beaker Homogenizer Polycarbonate centrifuge tube Ultracentrifuge Scaled polypropylene measuring cylinder Coarse cotton cloth Polypropylene funnel

Move

Materials and equipment

Chicken gizzards (25; stored at -20°C) (e.g., Pel-Freez, Inc.)

2-Mercaptoethanol (2-ME; molecular weight 78.13 Da; density 1.114)

Polypropylene beaker (4000-ml)

Homogenizer (e.g., BrinkmarmPolytron)

Polycarbonate centrifuge tubes (6x500-ml)

Ultracentrifuges, available with 3000-ml volume heads

Scaled polypropylene measuring cylinders (4000-ml)

coarse cotton cloth

Polypropylene funnel (large, wide mouth type)

Reagents

Buffer H (10X)

(For recipe, see "Preparation of Reagents" PP.82-83)

Operating Procedures

1) Take the gizzard and thaw it at room temperature for 2 h, then leave it at 4°C overnight.

2) Remove the white connective tissue between the two leaves of each gizzard with a scalpel, but do not worry about the small amount of connective tissue around the main leaf. Discard the connective tissue and cut the gizzards into approximately ^1cm3 pieces.

3) Weigh the cleaned pieces of gizzard tissue and record the weight.

4) Prepare 2L1x Buffer H by diluting 10x Buffer H Reservoir f with distilled water and add 70.1ul of 2-ME per L to make a final concentration of 1 mmol/L 2-ME.

5) Place the gizzard tissue in a 4000-ml polypropylene beaker and add two times the volume (i.e., 2 ml/g) of 1x Buffer H. For example, if 400 g of tissue is present, add 800 ml of buffer. In a cold room, transfer the buffered tissue to a homogenizer and homogenize three times at near maximum speed for 30 s each time.

6) Pour the homogenate into a 500-ml polycarbonate centrifuge tube and equilibrate it with a pasteurized pipette (the homogenate is quite viscous, so it is necessary to bend the tip of the pasteurized pipette to make the mouth of the pipette larger). Centrifuge the homogenate in a high speed centrifuge at 4°C for 30 min at 8000 r/min.

7) Pour the supernatant into a polypropylene funnel with two layers of cotton cloth and strain into a graduated polypropylene measuring cylinder. Allow the supernatant to flow along the side walls of the cylinder so that the extract does not foam profusely (as in the case of pouring beer to prevent foaming). Remove the precipitate with a spatula, combine, and return to the polypropylene beaker. Add 1x the volume of 1X Buffer H [based on the original wet weight of the raw material (see step 3 above)]. Repeat homogenization and centrifugation.

8) Combine the supernatants from the first and second runs and record the total volume. Leave 0.1% (approx. 1 ml) of the supernatant for subsequent activity determination. You will need to know these two volumes for calculations.

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