This experiment describes the trypsin digestion process of calmodulin. This experiment was derived from Protein Purification and Identification Laboratory Guide by Houzhu Zhu.

Operation method

Trypsin digestion assay of calmodulin

Materials and Instruments

Digestive buffer TPCK-treated trypsin HCl Pure calmodulin

Move

makings

TPCK-treated trypsin (dry powder available from WorthingtonBiochemicalCorp.)

HCl (1 mmol/L)

Pure calmodulin (dry powder; salt-free (0.1-1 mg)

Reagents

Digestive buffer

(For the recipe, see 'Preparation of reagents', PP.82-83)

Procedure

1) Weigh 0.1~1mg of trypsin in a 1.5-ml centrifuge tube and dissolve in 1mmol/LHCl to a final concentration of 1 mg/ml. place on ice.

2) Dissolve calmodulin in digestion buffer at a final concentration of 1 mg/ml on ice.

3) Add one aliquot of trypsin to calmodulin. The weight of trypsin in this aliquot should be 1/100 of the total amount of calmodulin. e.g., for 0.5 mg of calmodulin, add 5ug of trypsin (5ullmg/ml trypsin reservoir).

4) Cap the centrifuge tube tightly and incubate the reaction mixture at 37°C for 2 h. The reaction mixture should be incubated for 1/100 of the total amount of calmodulin.

5) While the digestion reaction is in progress, place the remaining trypsin reservoir on ice. 2 h later, add another aliquot of trypsin (1/100 of the weight of calmodulin) to the digestion mixture. Cover the tube tightly and continue incubation at 37°C for another 4-8 h (or overnight if possible).

6) At the end of the reaction, the product can be lyophilized to the desired level, vacuum centrifuged to reduce the volume, or injected directly into the sample for HPLC separation. The sample can be acidified to pH 2-3 with HC1 or trifluoroacetic acid to terminate the reaction. However, there is a risk that some peptides with low isoelectric points may precipitate.

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