1. What precautions should one take when using caged compounds? Top
Caged compounds are very sensitive to light, pH fluctuations, and heavy metal ions. At or below 360 nm the uncaging is accelerated. It is recommended that all experiments be performed in diffused light. Solutions of caged compounds should be aliquoted for single time use and stored in a freezer in a dark vial. The aliquoted stock solution should be dispersed into the experimental medium just prior to use. Any unused portion should be discarded.

2. What is the best way to eliminate precipitation of my fibrinogen sample? Top
It is important that solutions used for reconstituting fibrinogen be first brought to 37°C. Once at 37°C, the solution can be added to the fibrinogen and this mixture should remain at 37°C until the fibrinogen is completely solubilized.

3. How do I determine if a compound is cell-permeable? Top
There is no easy answer to this question. Generally charged molecules and phosphorylated intermediates such as glucose-6-phosphate; phosphoenolpyruvate etc) are not cell-permeable. However, modified phosphorylated compounds, such as mono- and dibutyryl cAMP, are cell-permeable. High molecular weight peptides and proteins are not cell-permeable under normal conditions.

4. What is the purpose of adding small quantities of proteins (BSA or HSA) to some of our growth factor and cytokine preparations? Top
Some products may have a tendency to stick to the sides of the vial. If the material is provided in very small quantities (micrograms), this may cause a large percentage loss. Addition of proteins, such as BSA, helps reduce the adherence to the vial. Also, the protein may be more stable in a solution with BSA or HSA in buffer alone.

5. Why does Calbiochem sometimes ship chemicals at room temperature when the vial is labeled 'Refrigerate' or 'Freeze'? Top
Storage in the refrigerator or freezer is recommended for 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 one should store it in refrigerator or freezer (as indicated on the label).

6. What is meant by "Sold on the basis of peptide content"? Top
Products labeled as "sold on the basis of peptide content" contain a specified amount of the peptide. For example, if the product is sold in 1 mg size and the peptide content is 90%, there will be about 1.11 mg of the total material in the vial (1 mg peptide plus stabilizers or salts). If you must make a 1 mg/ml solution, dissolve the entire content of this vial in 1 ml of solvent. Alternatively, if the product is sold by weight, there would be only 1 mg of material in the vial. Assuming an identical peptide content, the solution of the latter will have a concentration of 0.9 mg/ml.

7. What are Aquacides and how can they be used? Top
Aquacides are excellent material for rapid removal of water from dialysis tubing containing protein solutions. Aquacide I (Average M.W. 70 kDa) and Aquacide II (average M.W. 500 kDa) are carboxymethylcellulose based materials. Aquacide III is a polyethylene glycol based material Average M.W. 8 kDa). There are two common methods of using Aquacides: (a) Make a 2% syrup of aquacide and chill. Place the dialysis bag, with protein solution, in this syrup and weight down the dialysis bag. Place in a refrigerator for 6 to 8 hours. (b) Pack the dialysis bag, with protein solution, in aquacide and place in a refrigerator for 6 to 8 hours. Aquacides neither denature proteins nor change the pH during concentration. At room temperature 1 g of Aquacide absorbs about 5 ml of water from solution in one hour.

8. How much G418 should be used for selection of resistance? Top
The optimal concentration of G 418 for selection of resistance will vary according to the organism and/or cell type under investigation. In general, the concentration of active drug required for selection is as follows:

Dictyostelium sp.: 0.01 - 0.1 mg/ml Plant cells: 0.01 - 0.1 mg/ml Yeast cells: 0.5 - 1.0 mg/ml Mammalian cells: 0.1 - 2.0 mg/ml

A multiplying cell will be affected by the presence of G 418 sooner than a resting cell. It will take at least two cell generations to achieve cell death in sensitive cell lines.

9. How can one calculate concentration by spectrophotometric measurements? Top
As per 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 å (epsilon). å 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. å is also used to characterize compounds and establish their purity.

Example:

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

Hence, 5 mg/liter (0.005 g/l) read 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/liter. 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 A (1%, 1 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; where K = c/A. The value of the constant K is obtained by measuring the absorbance (A) of a standard of known concentration (c).

10. What is the advantage of using 7-amino-4-trifluoromethyl coumarin (AFC)-based substrates over 7-amino-4-methyl coumarin (AMC)-based substrates? Top
The AFC-based substrates offer several advantages over the AMC-based substrates. The Stoke's shift for the substrates with AFC groups is larger than those with AMC groups. This minimizes any spectral interferences. In addition, the trifluoro group is resistant to photobleaching. Photobleaching is a process wherein under high-intensity illumination conditions an irreversible destruction of the excited fluorophore is observed.

11. How much of an inhibitor or stimulator should one inject into an animal? Top
There is no simple answer to this question. One must optimize the dose empirically by performing a few preliminary experiments. First determine if the compound in question is cell-permeable. Also, survey the literature for any reported IC₅₀, ED₅₀, or EC₅₀, values. One may follow the sample calculation given below as a general guide:

H-89, dihydrochloride, a cell-permeable protein kinase A inhibitor, has an IC₅₀, value of 48 nM. It has a molecular weight of 519.3. In this case 240 to 480 nM range of H-89 is sufficient to cause maximal inactivation of protein kinase A. To use it in vivo we have to make a few assumptions. If a rat weighs about 200 g and we assume that 70% of its body weight is water, the volume of distribution will be approximately 140 ml. In this case 240 nM = 240 nmoles/liter = 124.63 mg/liter. We have assumed the volume of distribution to be about 140 ml. Hence, 124.63 x 0.140 = 17.45 mg would be the required amount for injection into the rat. It is important to note that the drug distribution will vary depending on the mode of injection (intravenous, intramuscular, or intraperitoneal), bioavailability, half-life, rates of hepatic and renal clearance, binding to proteins, and tissue-specific distribution and accumulation. The specific tissue uptake may also be limited in whole organs or tissues as compared to isolated cell preparations. In whole animal studies, sometimes a loading dose is required to achieve the target concentration. This may then be followed by a sustained infusion to maintain the drug level in the blood. One must always exercise caution and not overdose the animal.

12. How should one select a proper fluorochrome? Top
Fluorescence staining methods offer several advantages such as high resolution, live cell staining, and the possibility of dual labeling. Fluorescein and rhodamine are the two most commonly used fluorochromes. Fluorescein emits a yellow-green light that can be visually detected. However, it is prone to rapid photobleaching. Photobleaching can be retarded by using compounds such as DABCO. Rhodamine emits a red color, and it does not fade as quickly as fluorescein. Also, rhodamine conjugates are more hydrophobic and yield higher backgrounds than fluorescein. A third fluorochrome, TEXAS RED®, emits strong red light and does not fade easily. Its emission and excitation are at longer wavelengths. However, it is not as widely available as the other popular fluorochromes. A pair of fluorochromes, usually fluorescein and either rhodamine or TEXAS RED®, may be used on the same specimen. In such cases exercise catution in choosing reagents to ensure that there is no overlap between the emission spectra of the labeling detection reagents.