EGTA-containing buffer

The interaction between aequorin and a chelator must be carefully considered when estimating Ca2+ concentrations with aequorin in a calcium buffer containing EDTA or EGTA. This is particularly crucial when using a common calcium buffer system that contains a constant total concentration of a chelator in the buffer solutions of various Ca2+ concentrations in such a buffer system, a buffer of lower Ca2+ concentration contains a higher concentration of the free form of the chelator, resulting in an increased inhibition. 

Fig. 4.1.8 Influence of various calcium chelators on the relationship between Ca2 " concentration and the luminescence intensity of aequorin, at 23-25°C (panel A) in low-ionic strength buffers (I < 0.005) and (panel B) with 150 mM KC1 added. Buffer solutions (3 ml) of various Ca2+ concentrations, pH 7.05, made with or without a calcium buffer was added to 2 pi of 10 pM aequorin solution containing 10 pM EDTA. The calcium buffer was composed of the free form of a chelator (1 or 2mM) and various concentrations of the Ca2+-chelator (1 1) complex to set the Ca2+ concentrations (the concentration of free chelator was constant at all Ca2+ concentrations). The curves shown are obtained with 1 mM MOPS (A), 1 mM gly-cylglycine ( + ), 1 mM citrate (o), 1 mM EDTA plus 2mM MOPS ( ), 1 mM EGTA plus 2 mM MOPS ( ), 2 mM NTA plus 2 mM MOPS (V), and 2 mM ADA plus 2 mM MOPS (A). In the chelator-free buffers, MOPS and glycylglycine, Ca2+ concentrations were set by the concentration of calcium acetate. Reproduced with permission, from Shimomura and Shimomura, 1984. the Biochemical Society.

Fig. 4.1.14 Relationship between Ca2+ concentration and the initial light intensity of various recombinant semisynthetic aequorins and w-aequorin J (a semisynthetic natural aequorin made from isoform J). The curve number corresponds to the number of semisynthetic aequorin used in Table 4.1.4. A sample aequorin (3 (Ag) was in 3 ml of calcium-buffer solution containing 1 mM total EGTA, 100 mM KC1,1 mM Mg2+ and 1 mM MOPS (pH 7.0), at 23-24°C. From Shimomura etal., 1993a, with permission from Elsevier.

Fig. 7.4.3 Relationships between the total light emission of polynoidin and the concentrations of the photoprotein (A), H2O2 (B), Fe2+ (C), and EGTA (D). The basic buffer solution used was 50 mM phosphate buffer, pH 6.6, containing 3 mM H2O2, 0.1 mM FeSC>4 and 10 mM EGTA the concentrations of H2O2, FeSC>4 and EGTA were varied in B, C and D, respectively. FeSC>4 was added last to initiate light emission. From Nicolas et al., 1982, with permission from the American Society for Photobiology.

The frozen shells were ground in a cold mortar with 50 mM sodium acetate buffer, pH 5.8, containing 10 mM EGTA and 0.2 M NaCl, then the mixture was centrifuged. The pellets were re-extracted with the same buffer, and centrifuged. All supernatants were combined, and the photoprotein was precipitated with ammonium sulfate. The photoprotein in the precipitate was purified by four steps of column chromatography at near 0°C. Due to the instability of the photoprotein, efforts were made to reduce the time required for purification. 

Step 1. Gel filtration on Sephadex G-100, in 10 mM sodium phosphate buffer, pH 6.5, containing 5mM EGTA and 0.2 M NaCl, which was the basic buffer used throughout the purification process. 

Morgan et al. reported that Ki-67 immunostaining was completely suppressed when FFPE sections were heated in a solution containing CaCl2, but recovered when heated in buffers containing EDTA or EGTA, and proposed... 

CaM was purified from porcine brain. The purity of proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. CaM and PDE were cross-linked with l-ethyl-3(3-dimethylamino-propyl) carbodiimide (EDC) or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) in a buffer solution of 0.1 M HEPES (pH 7.1) in the presence of 1 mM CaCl2. After a buffer solution containing 2 mM EGTA added into the reaction solution, the CaM-PDE hybrid was separated from other ingredients by gel chromatography on a Sepharose CL-6B solumn. 

Protease treatment After washing, cells are exposed to a solution of 0.04% protease (pronase E) in Ca TMg free HEPES-containing ethylene glycol tetra acetic acid (EGTA) (Sigma) for 15 minutes at 37°C as applied in (93). Cells are then washed three times with ice-cold phosphate buffered saline (PBS) containing 0.1% BSA. 

Figure 38. The SPR signal intensities as a function of concentration of free Ca ions in 150 mM NaCl containing 0.5 mM EGTA and 10 mM HEPES buffer (pH 7.5). ...

The platelet pellet is resuspended in HEPES-Tyrode buffer containing 5 mM EGTA and 2 pM PGEi. 

The enzyme assay contained 0.1 mM 5,6-dihydroxyindole-2-carboxylic acid in 30 mM Tris-HCl buffer (pH 7.8), 2.5 mM MgCl2,1 mM 5-adenosylmethio-nine (as methyl donor), 0.5% Triton X-100,0.5 mM EGTA, and 5 mAf dithio-threitol in a total volume of 250 / L. The reaction was stopped with 25 fiL of 4 M perchloric add. Following centrifugation, the supemate was analyzed by HPLC. Product formation was linear with time for 60 minutes. 

Remove the Ringer solution from the synaptosomal pellet, resuspend the pellet in 200 fil acidification buffer containing EGTA and SLO (5fil/200nl, dilution depends on batch) or alpha-toxin (12-20[il/200[xl, dilution depends on batch). 

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