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The overall goal of this procedure is to synthesize, purify, and characterize lanthanide containing complexes that can be used for tagging biological macromolecules. This is accomplished by first mixing the metal starting material and ligand while controlling the pH of the solution. The second step of the procedure is to purify the resulting complex using dialysis.

Next, the absence of free metal needs to be verified. The final step of the procedure is the characterization of the properties of the complex that are relevant to imaging. Ultimately, results can be obtained that show that the synthesized complexes will behave as contrast agents through relax activity measurements.

This method can help answer key questions in the imaging field, such as whether the water coordination number of a contrast agent changes upon binding to a protein. Generally, individuals need this method will struggle because net carefully controlling the pH will result in rendering the LI and useless for bio education. Demonstrating the dialysis and activity measurements will be Buda and Sashi.

Graduate students from my laboratory To begin methation using lanthanide salts first dissolve one equivalent of ligand in water to produce a 30 to 265 millimolar solution. The ligand Paraiso Benzyl DTPA is used here at a concentration of 73 Millimolar. Next, adjust the pH of the ligand solution to between 5.5 and seven by adding one molar ammonium hydroxide.

In this video, 0.2 milliliters of the one molar ammonium hydroxide solution is used now dissolve one to two equivalents of lanthanide chloride in water to produce a solution with a concentration of five to 1000 millimolar. Either euro bium chloride or gadolinium chloride can be used in concentrations of 111 millimolar. Often an excess of metal is used to drive the ventilation to completion and consequently simplify purification.

Next, add each solution of lathan salts to the prepared solution of ligand while stirring. Now adjust the pH of the resulting reaction mixtures to between 5.5 and seven by adding 0.2 molar ammonium hydroxide. Here, a total of 0.5 milliliters of the 0.2 molar ammonium hydroxide solution is used to adjust the solution.

If the ligand being used contains acid sensitive functional groups, adjust the pH multiple times during this step. Be sure to use caution when adjusting the pH because if the solution becomes too basic, any base sensitive functional groups like Isothiocyanate will be rendered unusable. For conjugation, closely monitor the reaction via pH measurements.

The reaction is complete when the pH remains constant For ligands without any base sensitive functional groups, A workup that involves raising the pH may also be useful. To begin dialysis workup, determine an appropriate length of dialysis tubing to hold the sample volume plus extra length to hold an additional 10%of the sample volume. Then follow manufacturer guidelines to cut the tubing to this length.

In this video, a 100 to 500 Dalton molecular weight cutoff membrane was used, but larger molecular weight cutoff tubing can be used as appropriate if conjugation is performed prior to methation, if appropriate, based on the manufacturer's guidelines, soak the cut dialysis tubing, and water for 15 minutes at ambient temperature. Next, fill a dialysis reservoir with water, which will serve as the dialysate. A one liter beaker is used here.

The dialysate volume should be approximately 100 times that of the sample. Now fold one end of the tubing twice and secure the folded portion with the dialysis closure clamp. Wrap the end of the closure with a rubber band to ensure that it remains closed during dialysis.

Filter the previously prepared reaction mixture through a 0.2 micron filter. Then load the filtrate into the open end of the tubing. Being careful not to tear the tubing.

Be sure to leave enough head space to close the tubing. Fold the remaining open end of the tubing twice, then secure it with a closure and wrap the closure with a rubber band. Next, attach a glass vial containing air to the clamp on one end of the dialysis tubing using another rubber band.

Then attach a vial containing sand to the other clamp. These vials ensure that the tubing stays immersed in the dialysate. Now, place the full tubing in the dialysis reservoir that contains dialysate.

Stir the dialysate using a magnetic stir plate at a slow speed at ambient temperature. Be sure the stir speed remains slow and the solution does not.Vortex. Change the dialate three times over the course of a day.

In this video, the dialate is changed at 2.5, 6.5, and 11.5 hours. Allow dialysis to continue overnight for total of 20 to 28 hours. Once the dialysis is complete, remove the tubing from the dialate and carefully open one closure to remove the sample.

Wash the dialysis tubing three times with water and combine the washings with the sample. Finally, remove the water from the sample under reduced pressure here. This is accomplished by freeze drying.

The sample is frozen and then placed on a freeze drying apparatus. To assess the presence of free metal in the sample, first prepare acetate buffer by dissolving 1.4 milliliters of acetic acid in 400 milliliters of water, and adjust the pH to 5.8 with one molar ammonium hydroxide, and add water to produce a total volume of 500 milliliters. Then dissolve 0.3 milligrams of each complex in 0.3 milliliters of buffer.

Now prepare xol orange indicator, which should be 16 micromolar in the pH 5.8 buffer. Add three milliliters of this indicator solution to the previously dissolved metal complexes. Detect the presence of free metal via observation of a color change of the indicator from yellow to violet.

If desired, the amount of free metal can be quantified by creating a calibration curve. If free metal remains, the sample should be further purified using dialysis or high performance liquid chromatography prior to characterization. Next, to determine the water coordination number for a opium sample, prepare a solution of about one millimolar of the opium containing complex in water, and then another solution of the same concentration in deuterium oxide.

Prior to analysis, the deuterium oxide solution must be evaporated and dissolved in deuterium oxide three times to remove residual water, turn on the spectro fluorimeter, add the water solution to a clean vete and place the vete into the spectro fluorimeter. Now perform excitation and emissions to determine the maxima for each at approximately 395 nanometers, 595 nanometers respectively. Next, perform a phosphorescence time decay experiment using the excitation and emission wavelengths just determined and the parameters displayed here.

Repeat this step with a prepared deuterium oxide solution from the luminescence decay data just obtained plot, the natural log of intensity versus time. The slope of these lines are the decay rates. In this video, Microsoft Excel 2007 was used to generate the natural log plots from the raw data.

Use the decay rates in the equation developed by hors and coworkers seen here. If your ligand contains OH or NH groups coordinated to the metal, then the equation must be modified before use. To determine the relativity measurements of the sample first, select the desired application mode on the relaxation time analyzer, either T one or T two.

Here, the T one setting is selected. Next, prepare a series of samples that contain different concentrations of the lanthanide containing complex in an acquiesce solvent. Here, water is used as the solvent and solutions of ten five two 0.5, 1.25, 0.625 and zero.

Millimolar are prepared. Other acquiesce solvents or buffers can be used, but it is important to use the solvent as the blank. The final volume of the sample is specific to the instrument that is being used.

Place a sample in the instrument and let it sit for five minutes to equilibrate to the temperature of the instrument, which is 37 degrees Celsius for the unit used in this video. Now determine the relaxation time in units of seconds by adjusting the parameters of the software to obtain a smooth exponential curve for T one or T two. Repeat this for all samples, including the blank.

Now, calculate the inverse of the measured T one or T two relaxation values and plot. These values versus Lathan concentration and units of millimolar fit the plot with a straight line. The slope of the fitted line is the relax activity being R one or R two for T one and T two respectively.

Here we see the general scheme for mediation and purification. This scheme depicts the general procedure for methation and reasons for choosing different purification roots. Here we see a representative luminescence decay plot in which the natural log of decay is plotted versus time the slopes of the lines generated from similar curves required for water and deuterium oxide solutions are used with equation one to determine the water coordination number of opium containing complexes.

A representative example of livity measurements can be seen here with a slope of the fitted line being the sample's T one livity. In addition to the water coordination number and livity characterization described in the protocol, it is also important to characterize final products using standard chemical techniques. The identity of the compound can be obtained using mass spectrometry representative mass spectra showing the diagnostic isotope patterns for gadolinium and opium containing complexes are shown here.

Following this procedure, other methods like endr spectroscopy HPLC and elemental analysis can be performed to further characterize the resulting complexes. After watching this video, you should have a good understanding of how to synthesize, purify, and characterize lanthanide containing complexes that can be used for tagging biological macromolecules.