Technical Tips: Solubility/Media  |
1. What type of solvent is best suited for dissolving a compound? Top
In biological experiments water is the most preferred solvent. However, several organic compounds are either not soluble in water or they degrade rapidly in the presence of moisture. If DMSO is a recommended solvent it is best to use a fresh stock bottle of DMSO that is deemed free of any moisture. Any contaminating moisture may accelerate the degradation of compound in question or may render it insoluble.
2. Why can't I make serial dilutions of my DMSO stock solution directly in my buffer? Top
In some cases this may not be a problem. However, in most cases the organic material will precipitate out of the solution. It is best to make the initial serial dilutions only in DMSO and then add the final diluted sample to your buffer or the incubation medium. Also, the compound may be soluble in aqueous medium only at its working concentration.
3. What type of cell culture medium is suitable for my experiment with Calbiochem's nitric oxide or nitric oxide synthase assay kits? Top
Culture media such as RPMI 1640, Dulbecco Modified Eagle Medium (DMEM), and Media 199 contain high levels of nitrate. It is best not to use these type of media, particularly when small changes in nitrate levels are measured. If it is absolutely necessary to use this type of medium, then cellular nitrate/nitrite levels can be quantitated by subtracting the level of nitrate/nitrite in the medium (in the absence of cells) from the total levels recorded. Media such as Waymouth's MB 752/1 and minimum essential medium (MEM) are devoid of nitrates and may be suitable for use when assaying for nitrates and nitrites. However, we strongly recommend a pilot experiment to determine the suitability of any medium for your particular type of experiment.
4. What is the best method to reconstitute fibronectins? Top
Fibronectins are very sensitive materials. Be very careful during the thawing process. Place the vial in a 37ºC water bath and leave it undisturbed until it is completely thawed. Do not disturb or remove the vial at any time during the thawing period. If the vial is disturbed or removed prior to complete thawing, the product may gel and will be unusable. After thawing do not vortex or shake. Mix very gently.
5. What precautions should be taken during peptide solubilization? Top
Most peptides, when stored at -20°C, will remain stable for several years. When you are ready to use the peptide, first bring the vial to room temperature in a desiccator. Peptides containing Cys, Met and Trp may require special precautions to avoid any oxidation. Peptides should be dissolved in distilled water, dilute acetic acid, or other appropriate solvent stored in a tightly sealed bottle. Most peptides have a limited life in solution and long-term storage should be avoided. Buffer or saline should be added only after the peptide is fully in solution. If complete solubilization is not achieved solution can be mildly sonicated. Solutions should be aliquoted and stored in the pH range of 5 - 7 at -20°C. Any unused portion of the thawed aliquot should be discarded.
|
