EGTA Usage & Applications

EGTA (also, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ethylene glycol tetraacetic acid, or egtazic acid) is an aminopolycarboxylic acid and a strong calcium-specific chelator. EGTA is commonly used to remove free calcium ions (Ca2+) from solutions where the presence of calcium may be problematic. EGTA is related to EDTA, however has a much lower affinity for other metal ions like those from Mg, Mn, Co, Zn, Pb, Cu, and Fe, and is, alternatively, highly selective for Ca2+. This feature of EGTA is particularly useful in buffered systems that aim to resemble the environment of living cells, where some metal ions like Mg2+ are usually present in at least a thousandfold higher concentration than Ca2+.

EGTA has a number of uses across multiple fields of science, research and development. EGTA can be added to cell culture media to chelate contaminating Ca2+. Reducing Ca2+ helps to maintain sample viability and allows researchers to focus and isolate calcium-dependant signaling pathways. As a chelator, EGTA also has the potential to inhibit activity on other cation-dependent enzymes in culture.

The use of EGTA extends to enzymatic assays, which require Ca2+ as cofactors for activation. In enzyme reaction buffers, EGTA can efficiently remove Ca2+ to enable researchers to study the effects of calcium on enzyme activity. In such reactions, the amount of free Ca2+ can be calculated from determining the association constant of EGTA.

Common EGTA Stock Solution Preparation

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Volume of DMSO needed to reconstitute specific mass of EGTA to the given concentrations. These volumes are only for preparing the stock solution, and researchers should consult their specific experimental protocols for buffer requirements.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	149.566 µL	747.831 µL	1.496 mL	7.478 mL	14.957 mL
5 mM	29.913 µL	149.566 µL	299.133 µL	1.496 mL	2.991 mL
10 mM	14.957 µL	74.783 µL	149.566 µL	747.831 µL	1.496 mL

Sample Protocol for Calcium Measurement

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Titrate the concentration of free Ca²⁺ in solution by mixing graduated amounts of K₂EGTA and CaEGTA. The reactions of these solutions with a fluorescent calcium dye (such as Fluo-3, Fluo 4, Fluo-8®, Cal-520®, or Calbryte™ 520) should be at room temperature, pH 7.2 and 100 mM KCl.



Measure the dye fluorescence intensity with a fluorescence microplate reader at Ex/Em = 490/525 nm.

1. Mix the relative volumes of EGTA tetrasodium salt (K₂EGTA) and CaEGTA according to the following table:
   
   

   Sample	Volume K2EGTA, uL	Volume CaEGTA, uL	Calculated free Ca2+, uM	RFU
   zero (blank)	1000	0	0	0
   1	900	100
   2	800	200
   3	700	300
   4	600	400
   5	500	500
   6	400	600
   7	300	700
   8	200	800
   9	100	900

   
   
   
2. Calculate the concentration of free Ca²⁺ in each solution using the following formula:
   
   [Ca²⁺]_free = K_d^EGTA x {[CaEGTA]/[K₂EGTA]}
   
   
3. Add 1 uL of 1 mM fluorescent calcium indicator into each solution including the blank.
4. Read the fluorescence intensity of each solution with a fluorescent microplate reader at Ex/Em = 490/525 nm.

Other Uses

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EGTA is also a common ingredient in some lysis buffers, like RIPA for DNA extraction, to prevent degradation by calcium-dependent nucleases. EGTA, along with other additives in lysis buffers, aid in protein solubility, stability, and play a critical role in protein quality and yield downstream. EGTA can be utilized to deactivate enzymes, like DNase, that need metallic cofactors for catalytic activity.

EGTA and similar chelators may also play a part in protein purification to remove Ca2+, thereby preventing unwanted protein-protein interaction or problematic protein aggregation. In tandem affinity purification and similar methods, EGTA is commonly added to the elution buffer to help dissociate proteins bound to the solid phase matrix.

In research, EGTA has shown to improve the accuracy of electrophysiological reactions. In particular, EGTA has found use in patch-clamp and multi-electrode array electrophysiology techniques that aim to measure the dynamic functional properties of neurons. These studies help reveal cell-specific synaptic and cellular conditions that govern neurotransmission. In the process, EGTA is added to solutions to prevent calcium-dependent inactivation of voltage-gated calcium channels.

In dentistry research and endodontic therapies, EGTA is generally accepted as the most effective compound for preparing teeth prior to a root canal. Preparation of the root canal involves removal of a “smear” layer, a layer of material composed of dentine, pulp tissue, and occasionally bacteria that forms on canal walls. For better adhesion of filling materials to the root canal, this smear layer must first be removed. Both EDTA and EGTA have been used in these studies, though EGTA has shown to effectively remove the smear layer without eroding underlying dentinal tubuli, like EDTA does.

Resources:

Bath Solution (Ca2+/Mg2+ Free) Preparation and Recipe Chelation Medium Preparation and Recipe MOPS-EGTA buffer Preparation and Recipe

Products

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References

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The Effects of EGTA on the Quality of Fresh and Cryopreserved-Thawed Human Spermatozoa
Cation chelators and their utilization in the preparation of low concentrations of calcium. Caution of use in biological systems with high affinity to calcium
Lysis Buffer
Smear layer removal by EGTA
CHEBI:30740 - ethylene glycol bis(2-aminoethyl)tetraacetic acid