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9 th PBL in Calcium and Phospholipid Signaling Department of Clinical Science and Education; PowerPoint PPT Presentation


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9 th PBL in Calcium and Phospholipid Signaling Department of Clinical Science and Education; Department of Physiology and Pharmacology. May 4-15, 2009. Md. Shahidul Islam, M.D., Ph.D. Associate Professor Department of Clinical Research and Education Karolinska Institutet

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9 th PBL in Calcium and Phospholipid Signaling Department of Clinical Science and Education;

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9 th pbl in calcium and phospholipid signaling department of clinical science and education

9 th PBL in

Calcium and Phospholipid Signaling

Department of Clinical Science and Education;

Department of Physiology and

Pharmacology. May 4-15, 2009

Md. Shahidul Islam, M.D., Ph.D.

Associate Professor

Department of Clinical Research and Education

Karolinska Institutet

Forskningscentrum, Södersjukhuset

118 83 Stockholm, Sweden

[email protected]


Preparation of multiple ligand ion solutions

[L.Ca]

Ka

=

[L].[Ca2+]

Preparation of multiple ligand-ion solutions

Association constant or binding constant

L + Ca2+

L.Ca


Dissociation constant

[L].[Ca2+]

Kd

=

[L.Ca]

At half saturation of the ligand: [L]=[L.Ca]

And Kd = [Ca2+]

Dissociation constant

Kd=1/Ka


There are many ca 2 buffers to choose from

There are many Ca2+ buffers to choose from

  • EGTA

  • BAPTA

  • Dibromo BAPTA

  • Quin 2

  • Fura-2

  • EDTA


Affinity of the ligand for ca 2 depends on

Affinity of the ligand for Ca2+ depends on

  • pH

    • EGTA more than BAPTA

  • Temperature

  • Ionic strength =0.5SCi.׀Zi׀


How do i know which buffer to use

How do I know which buffer to use?

  • Kd should be close to the desired [Ca2+]

  • Affinity for Ca2+ must be much higher than Mg2+

  • pH-sensitivity of affinity for Ca2+

  • Costs etc


9 th pbl in calcium and phospholipid signaling department of clinical science and education

Ca2+ buffers exist in multiple states of protonation

[Lt]-[CaL]=[L]+[HL]+[H2L]+[H3L]+[H4L]

At pH 6 to just over 7, 99% of EGTA is in the form of H2EGTA

Only two forms of EGTA, EGTA4- and EGTA3- bind Ca2+


9 th pbl in calcium and phospholipid signaling department of clinical science and education

Some important properties of Ca2+ buffers

Ca2+ buffer

Kd

KCa/KMg

EGTA

67 nM

6.2

72.2

KCa(pH 7.4)

(pH 7.4)

BAPTA

192 nM

1.14

158.24

KCa(pH 7.0)

DiBromo

BAPTA

1.83 mM

1.02

63.00


9 th pbl in calcium and phospholipid signaling department of clinical science and education

EGTA is the ”devil we know”

  • Kd near intracellular [Ca2+]i

  • 100,000 times higher affinity for Ca2+ over Mg2+

  • Marked pH-dependence of Ca2+-affinity

  • Variable purity of EGTA

  • 30 times cheaper than BAPTA

  • EGTA itself may have non-specific effetcs

  • on processes studied


9 th pbl in calcium and phospholipid signaling department of clinical science and education

A. Multiple Ligand Approach

When solutions contain more than one ligand

Use computer programs

Two methods of making Ca2+ buffers

B. Two solutions approach

Mix two solutions in various proportions

Single ligand

Ca2+ calibration solutions


9 th pbl in calcium and phospholipid signaling department of clinical science and education

Making of Ca2+ buffers

  • Use High Purity Water

  • Use high purity EGTA, BAPTA

  • Use Plastic Wares

  • Use Ca2+ standard solutions

  • And not CaCl2.2H2O

  • Accuracy in measurement of pH

  • Check by Ca2+ eletrodes


9 th pbl in calcium and phospholipid signaling department of clinical science and education

Lowering Extracellular [Ca2+]

A. Reducing to <1 mM

Stock solution of 1 M Na2H2EGTA

(pH 7.4)

Add Na2H2EGTA three times the concentration

Of Ca2+

B. Reducing Ca2+ to 0

Nominally Ca2+-free medium

Add Na2H2EGTA, 1 mM

C. Precisely known concentration of Ca2+

Use computer programs

Check with Ca2+ electrodes

Or use two solutions approach


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