Team:Korea U Seoul/Project/sub proc result

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CaCO3 Precipitation Assay
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CaCO3 Precipitation Assay

Method

The overall reaction for precipitation is
Ca2+ + HCO3 - → CaCO3 + H+
According to that equation, CaCO3 generated the more the pH will be lowered.

The effect of NA and R on the rate of precipitation of CaCO3 was determined by recording the decrease in pH of a 20mM Tris HCl pH8.0 buffer solution at 10°C


Reagents

  • 20mM Tris-HCl buffer, pH 8.0.
  • 20mM Tris-HCl buffer, pH 8.0 + 1M Imidazole.
  • 1M NaHCO3.
  • 1M CaCl2.


Enzyme

Using Protino® Ni-NTA Columns for His-tag protein purification, purify the enzymes.

These enzymes is diluted in 20mM Tris-HCl buffer, pH 8.0 + 10mM Immidazole at 4°C.


Procedure

- Blank Determination Add 0.5 ml of 1M CaCl2 to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Immediately start a stop watch and record the time and pH up to 20 minutes. - Enzyme Determination Add freshly diluted enzyme to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Add Add 0.5 ml of 1M CaCl2. Immediately start a stop watch and record the time and pH up to 20 minutes.

Contents

Method

               The overall reaction for precipitation is
Ca2+ + HCO3 - → CaCO3 + H+
               According to that equation, CaCO3 generated the more the pH will be lowered.


               The effect of NA and R on the rate of precipitation of CaCO3 was determined by recording the decrease in pH of a 20mM Tris HCl pH8.0 buffer solution at 10°C

Reagents

  • -20mM Tris-HCl buffer, pH 8.0.
  • -20mM Tris-HCl buffer, pH 8.0 + 1M Imidazole.
  • -1M NaHCO3.
  • -1M CaCl2.

Enzyme

               Using Protino® Ni-NTA Columns for His-tag protein purification, purify the enzymes.


               These enzymes is diluted in 20mM Tris-HCl buffer, pH 8.0 + 10mM Immidazole at 4°C.

Procedure

               - Blank Determination
               Add 0.5 ml of 1M CaCl2 to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Immediately start a stop watch and record the time and pH up to 20 minutes.
               - Enzyme Determination
               Add freshly diluted enzyme to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Add Add 0.5 ml of 1M CaCl2.  Immediately start a stop watch and record the time and pH up to 20 minutes.


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