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Technical Tips
 | | | | | | |   How much inhibitor should I use? | | The amount of inhibitor required depends on various factors, such as target accessibility, cell permeability, duration of incubation, type of cells used, etc. It is best to survey the literature to determine the initial concentration. If published Ki or IC50 values are known, one should use 5 to 10 times higher than these values to maximally inhibit enzyme activity. If Ki or IC50 values are unknown, then one should try a wide range of inhibitor concentrations and use Michaelis-Menten kinetics to determine the Ki value. It is not unusual to see either no inhibition or even a reverse effect when high concentrations of inhibitors are used. Researchers should always run an appropriate control to eliminate non-specific effects of the solvent used to solubilize the inhibitor. | | | | What is the difference between EC50, ED50, Ki, IC50, and Kd? | In pharmacology and biochemistry, in order to determine the efficacy of a drug or inhibitor, the following terms are commonly used. Sometimes, confusion arises when researchers try to repeat experiments without considering the exact term used by the original investigators.
EC50: Clinical efficacy of a drug (concentration required) to produce 50% of the maximum effect (may be inhibitory or stimulatory effect). This term is used usually with pharmaceuticals.
ED50: Median effective dose (as opposed to concentration) at which 50% of individuals exhibit the specified quantal effect.
IC50: Concentration required to produce 50% inhibition.
Ki: Inhibitor concentration at which 50% inhibition is observed (it is calculated using Michaelis-Menten kinetics).
Kd: An equilibrium constant for the dissociation of a complex of two or more biomolecules into its components; for example, dissociation of an inhibitor or substrate from an enzyme. | | | | How much of an inhibitor or stimulator should one inject into an animal? | 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. For H-89, 2HCl a 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. Because the volume of distribution is about 140 ml, 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. | | | | 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 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. | | | | 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 when added directly to an aqueous medium. It is best to make the initial serial dilutions only in DMSO and then add the final diluted sample to your buffer or the cell culture medium. Also, the compound may be soluble in aqueous medium only at its working concentration. | | | | Which protein kinase inhibitor is best suited for my experiment? | | If the mechanism involved in phosphorylation is unknown, a broad range inhibitor, such as Staurosporine, should be used first to determine if indeed a protein kinase is involved. Secondly, a more specific inhibitor of PKA (e.g., H-89, CN 371963, or 8-Br-cAMP, Rp isomer, CN 116816), PKC (e.g., Bisindolylmaleimide, CN 203290), or PKG (e.g., KT5823, CN 420321; or PKG inhibitor, CN 370654) should be used to eliminate the possibility of more than one kinase. To elucidate the exact mechanism involved, isozyme specific inhibitors, such as for PKC isozymes, can be used. | | | | How can I determine if a caspase inhibitor is reversible or irreversible? | | The C-terminal group determines the reversibility or the irreversibility of any caspase inhibitor. In general, caspase inhibitors with an aldehyde (CHO) group are reversible. The CMK, FMK, and FAOM groups are more reactive and form covalent bonds with the enzyme, creating an irreversible linkage. FMK is slightly less reactive than CMK and therefore is considered more specific for the enzyme site being inhibited. | | | | What determines the specificity of a particular caspase inhibitor? | | The peptide recognition sequence determines the specificity of the inhibitor for a particular caspase. Sometimes the aspartic acid residue is esterified to increase cell permeability of the peptide. VAD is a general caspase inhibitor. Earlier it was considered to be specific for caspase-1 (ICE), however, now it is considered to inhibit even caspase-3 and caspase-4. Addition of a tyrosine residue (Y) to the sequence (YVAD) makes the inhibitor more specific for caspase-1. The sequence DEVD recognizes caspase-3 and also caspases-6, -7, -8, and -10. | | | | What are the advantages of using FMK based caspase inhibitors and how do they differ from CHO-based inhibitors? | The FMK-based caspase inhibitors covalently modify the thiol group of the enzyme making them irreversible inhibitors. Generally, at the amine end of the inhibitor we have a benzyloxycarbonyl (Z), biotin, or aceytl (Ac) group. These groups also increase hydrophobicity of the molecule, which makes them more cell-permeable. Compared to the inhibitors with an Ac or a biotin group, those inhibitors with a Z-group are even more cell-permeable. Inhibitors with a biotin group can serve as a detection tool and are useful in tagging the enzyme-inhibitor site.
The CHO-based inhibitors are reversible due to the fact that the thiol group of the enzyme forms an adduct to the carbonyl group of the aldehyde that is reversible. As a general rule CHO-based inhibitors are hydrated and hence are slow binding. The extent of their reversibility depends on the pH, metal ion concentration, and other conditions. When the aldehyde group is attached to the aspartic acid (D-CHO), the product exists as a pseudo acid aldehyde in equilibrium. This makes it somewhat cell-permeable. | | | | What criteria should I use when selecting a protease inhibitor? | | When processing cells or tissues one must assume that active proteases are present in the medium or are being secreted. Hence, it is important to include protease inhibitors even in the early steps of sample preparation. For best results add protease inhibitors to the medium just prior to use. Use of inhibitors in buffers stored over a period of time is not recommended. Different cells and tissue types exhibit different protease profiles. Serine proteases are widely distributed in all cells, bacterial cells contain higher levels of serine and metalloproteases; animal tissue extracts are rich in serine-, cysteine-, and metalloproteases, and plant extracts contain higher quantities of serine and cysteine proteases. If you are not sure of the type of proteases present in the sample, it is best to use an inhibitor cocktail available from Calbiochem or customize your own cocktails. | | | |
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