This experiment describes the process of calreticulin sample activation measurement. This experiment is from the Experimental Guide for Protein Purification and Identification, by Hauchu Zhu.

Operation method

Activation assay of calmodulin samples

Materials and Instruments

Substrate peptide for MLCK ATP Calmodulin reservoir MLCK enzyme Phosphate Acetone (optional) MLCK assay buffer
Polypropylene microcentrifuge tubes WhatmaundefinedP81 Cellulose phosphate paper (cut into strips) Glass beakers Sampler Scintillation vials Scintillation counter Blow dryer (optional)

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Materials and equipment

Polypropylene microcentrifuge tubes (1.5-ml)

Substrate peptide for MLCK (10 mmol/L concentrated reservoir solution; for activity measurement, dilute 1:20 with water to 0.5 mmol/L solution)

ATP (2 mmol/L reservoir, containing 5 μCi of [ ^γ-32P ]ATP/5ul)

Calmodulin stock solution (0.1ug/ml or 1ug/ml)

MLCK enzyme (concentrate stored at -70°C; for activity measurements, dilute 1mg/ml bovine serum albumin 1:200)

Water bath

WhatmaundefinedP81 cellulose phosphate paper (cut into strips)

Phosphoric acid (75 mmol/L) (3X4L)

Glass beaker (3~4L volume)

Sampler (3~10-ml)

Scintillation bottle

Scintillation counter

Acetone (optional)

Blow dryer (optional)

Reagents

MLCK activation buffer (10x)

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

Operating Procedures

1) For each activation, place the following reagents in a 1.5-ml polypropylene microcentrifuge tube.

MLCK Activation Buffer (10x) 5ul

MLCK substrate peptide (0.5 mmol/L) 5ul

[ ^γ-32P ]ATP (2 mmol/L 35uCi/5ul) 5ul

Calmodulin (0.1ug/ml or 1ug/ml) or unknown samples from each purification step 0~20ul

Milli-Q water 10~30ul

MLCK enzyme (reservoir solution diluted 1:200) 5ul

Total volume 50ul

Note: Fractions from each purification step should be made up to an excess of 1 _mmol/LCaCl2 solution if they contain EDTA or EGTA. Several dilutions of each fraction should be measured, such as 1:10, 1:100, and 1:1000. Under the above conditions, the half effective dose of calmodulin is about 4 ng. The standard curve of calmodulin (0.2-20 ng) for the MLCK test is shown in Figure 1-2.



2) Start the experiment by adding MLCK enzyme. Incubate at 25°C for 20 min.

3) Place centrifuge tubes on ice. Remove a 10-ul aliquot from each tube and place on a small area of P81 paper. A strip of P81 paper can be used, and serial numbers (corresponding to the serial numbers of the centrifuge tubes) can be written on it in pencil at 2-3 cm intervals.

4) The strip is washed three times with 75 mmol/L phosphoric acid, manually agitated with a pipette, each time in a glass beaker, and the strip is transferred with a pipette. The solution from the first wash should be placed in the radioactive waste solution. After washing and blow-drying, the strips are cut into small pieces and placed in a scintillation vial for counting. Alternatively, the strips can be washed in acetone and blown dry quickly with a hand-held dryer (Marsh and Carroll, 1991).

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