FAQs on Inhibitor Preparation

Proper inhibitor preparation is key to the success of small molecule experiments. When reconstituting inhibitors in a solvent, short-term and long-term storage and stability should be considered. Some inhibitors may lose their potency quickly in aqueous solutions. In general, most organic molecules, but not all, can be dissolved and stored in dimethylsulfoxide (DMSO). However, always check the product data sheet or contact our technical service scientists for more information.

Here are a few frequently asked questions (FAQs) on inhibitor preparation to consider:

• What type of solvent is best suited for dissolving a compound?
• Why can’t I make serial dilutions of my DMSO stock solution directly in my buffer?
• How can I prevent my inhibitor in DMSO from precipitating in aqueous medium?
• What precautions should be taken during peptide solubilization?
• What precautions should be taken during peptide quantitation?
• Why are some small molecules shipped at room temperature when the vial is labeled as ‘Refrigerate’ or ‘Freeze’?
• How can you calculate concentration by spectrophotometric measurements?
  

What type of solvent is best suited for dissolving a compound?

In biological experiments, water is the most preferred solvent. However, several organic compounds are either insoluble in water or 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 the compound in question or may render it insoluble.

Why can’t I make serial dilutions of my DMSO stock solution directly in my buffer?

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. The compound may be soluble in an aqueous medium only at its working concentration.

How can I prevent my inhibitor in DMSO from precipitating in aqueous medium?

Some organic products dissolved in DMSO may fall out of solution when added directly to an aqueous solution, such as buffer or cell culture medium. To avoid this, dilute the concentrated stock solution further in DMSO before adding it to the aqueous medium. Most cells can tolerate up to 0.1% final DMSO concentration. Of course, a control with DMSO alone should always be included in the experiment.

What precautions should be taken during peptide solubilization?

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 cysteine, methionine, and tryptophan 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, the 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.

What precautions should be taken during peptide quantitation?

Determining peptide concentrations accurately and quickly can be difficult. Most commonly used methods for peptide quantitation rely on the weight of the lyophilized powder, the absorbance of ultraviolet (UV) light, or amino acid analysis. Establishing peptide concentration based on the weight of the lyophilized peptide is inaccurate in most cases because the analyzed powder can contain a significant quantity (10-70%) of bound water, salts, or counterions.

Another peptide quantitation method relies on absorbance at 280 nm, and thus can only be used to estimate peptide concentration if tryptophan and tyrosine residues are present in the sequence. Therefore, peptides that do not contain amino acids that absorb light at 280 nm cannot be accurately quantified using this method. While it is possible to determine peptide concentration by measuring absorbance at 205 nm, this measurement is far more sensitive to variations in sample composition, since many solvents and other chemicals will absorb at this wavelength.

Finally, amino acid analysis, recognized as a gold standard in peptide quantitation, delivers possibly the most accurate peptide quantitation; however, it is expensive and requires time-consuming sample manipulation along with specialized equipment.

Why are some small molecules shipped at room temperature when the vial is labeled as ‘Refrigerate’ or ‘Freeze’?

Storage in the refrigerator or freezer is recommended for the long-term stability of the product. If the material is shipped at ambient temperature it is considered to be stable for the duration of shipping and normal handling. Upon arrival, store the product in the refrigerator or freezer (as indicated on the label).

How can you calculate concentration by spectrophotometric measurements?

Sometimes it is critical to establish the exact concentration of a molecule in solution. The following example will be helpful not only in determining the concentration, but also the purity of molecules.

The relationship of absorbance to concentration is given by Beer’s law, A = abc where:

• A = absorbance
• a = a proportionality constant defined as absorptivity
• b = light path in cm
• c = concentration of the absorbing compound

When b is 1 cm and c is expressed in moles/liter, the symbol a is substituted by the symbol ε (epsilon). ε is a constant for a given compound at a given wavelength under prescribed conditions of solvent, temperature, and pH, and is referred to as molar absorptivity. ε is also used to characterize compounds and establish their purity.

Example Calculation

The molar absorptivity (ε) of bilirubin (Mol. Wt. = 584) dissolved in chloroform at 25 °C is 60,700.

Hence, 5 mg/L (0.005 g/L) of 100% pure bilirubin analyzed in a 1 cm cuvette should have an absorbance of A = (60,700)(1)(0.005/584) = 0.52 {A = abc}

Therefore, a solution of 5 mg/mL showing absorbance of 0.49 should have a purity of 94% (0.49/0.52 x 100).

In most biochemical and toxicological work, it is customary to list constants based on concentrations in g/dL rather than mol/L. This is also common when the molecular weight of the substance is not precisely known.

Here, b = 1 cm; and c = 1 g/dL (1%), and A is written as A¹% _cm) This constant is known as absorption coefficient.

The direct proportionality between absorbance and concentration must be established experimentally for a given instrument under specified conditions. Frequently there is a linear relationship up to a certain concentration. Within these limitations, a calibration constant (K) may be derived as follows: A = abc.

Therefore, c = A/ab = A x 1/ab. The absorptivity (a) and light path (b) remain constant in a given method of analysis. Hence, 1/ab can be replaced by a constant (K). Then, c = A x K and therefore K = c/A. The value of the constant K is thus obtained by measuring the absorbance (A) of a standard of known concentration (c).

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