Protein and Enzyme Activity Assays Support—Getting Started

Direct assays: The substrate or assay reagent is directly modified by the enzyme or directly interacts with the protein and signal is generated without the use of any intermediate reagent (excluding stop reagents) or enzyme reaction. Examples of a direct assay include our: EnzChek™ Protease Assays, Image-iT™ LIVE Poly Caspases Detection Kits, and CellEvent™ Caspase-3/7 Green Detection Reagent.

Indirect assays I: The substrate or assay reagent is directly modified by the enzyme or directly interacts with the protein but the signal is generated by interaction with another reagent or another reaction. The other reagents or reactions may be the second, third or even forth reaction from the initial enzyme reaction. Examples of an indirect assay I include our: Z’LYTE™ Activity Assays, LanthaScreen™ Kinase Activity Assays, and Adapta Universal Kinase Assay.

Indirect Assay II: The enzyme or protein generates a product which modifies a substrate or reagent through another enzyme/protein system and signal is generated either directly by this secondary reaction or by yet another enzyme or chemical reaction. Examples: Amplex™ Red Assays.

We offer various assays that allow for detection by absorbance (colorimetric), fluorescence or luminescence (bio- or chemiluminescence). Some reagents can be read in either absorbance or fluorescence mode, e.g. Amplex Red Assay. 

The first consideration in using a specific mode of detection is availability. What modes of detection are available for an assay? Some assays are not available in one mode or another due to the chemistry of the reaction or availability of reagents for the reaction.

Secondly, does the experimental sample have color (absorbance) or autofluorescence? Some natural materials are naturally colored or fluorescent. Examine your samples to see if they have a strong absorbance or absorbance in broad range. This may limit the ability to use a colorimetric assay. If interested in a fluorescence-based assay, examine your samples, media, etc. under all filters sets or channels for autofluorescence (fluorescence that is inherent to the sample due to various natural components), e.g. fluorescent proteins (GFP, etc.), porphyrins, dyes in media, some vitamins, natural pigments, etc. If a sample has high autofluorescence in a broad range of wavelengths, a fluorescence-based assay may not be compatible.

Time-resolved FRET (TR-FRET) and luminescence-based assays are desirable in that sample absorbance and autofluorescence can be avoided in the detected signal. TR-FRET involves exciting the sample with defined wavelengths, turning the excitation light off and then reading emission within 50 to 100 µsec after the light has been turned off.

Luminescence-based assays, either bioluminescent or chemiluminescent assays, do not require that a sample be exposed to any excitation light; light is generated by the biological or chemical modification of the substrate.

Ratiometric assays reduce the effects of well-to-well and day-to-day variations.

The detection range depends on the instrument and instrument settings. 

For the instrument, this is defined by the optics, the type of light source (for absorbance or fluorescence-based detection) and detector. Some microplate readers allow the adjustment of gain, i.e., the ability to place lenses or the sample closer to the detector, to increase the amount of light captured. 

Light sources may be lamps, LEDs, or lasers. As an excitation source, lamps may provide a broad range of wavelengths but may vary at certain wavelengths due to the inherent properties of the gas used in the lamp. LEDs may also provide a broad range of excitation wavelengths, but usually not as broad as lamps, hence an instrument may have to be equipped with more than one LED to accommodate a desired range of excitation wavelengths. Lasers emit at a very narrow range of wavelengths, for versatility, the instrument would have to be equipped with multiple lasers.

Detectors (the photomultiplier tube, ‘PMT’) vary in sensitivity based on their construction, materials, and design. Very sensitive detectors are required for assays using fluorescence polarization. Adjusting the voltage on the PMT changes the sensitivity of the detector. Increasing voltage increases sensitivity, but limits detection at higher signal. Decreasing the voltage decreases sensitivity to detect a greater amount of signal, but sensitivity may be lost. 

In adjusting instrument settings to optimize detection, you can adjust the gain, the intensity of the light source, and the voltage of the PMT to make the instrument more sensitive (for detecting at lower limits) or less sensitive to capture more signal.

As a general rule of ranges of sensitivity*:

Absorbance: nmole to µmole range/well (fluorescein, pH 9)

Fluorescence: fmole to µmole range/well (fluorescein, pH 9)

Luminescence: fmole to µmole range/well (ATP – Luciferin/luciferase)

* ranges may vary depending on the instrument. 

You may be limited by the instrument(s) available and the instrument’s ability to do certain types of detection or range of detection. Do not attempt to perform an assay on an instrument that is not equipped to provide the required mode of detection. 

You may be also limited by the inherent properties of your experimental samples. Is the sample highly colored or autofluorescent? If yes, then TR-FRET or luminescence may be the best mode of detection.

As long as the instrument provides the ability to detect in the mode required for the assay (absorbance, fluorescence or luminescence) and has sufficient sensitivity, it can be used. 

Other factors to consider, what types of microplates can or cannot be used with the instrument and whether the instrument has an auto-dispensing option. Microplate readers with automatic multichannel dispensing systems are important for kinetic assays or any other assay that measures a rapid flux (e.g., ion channels).

If purchasing a microplate reader, determine what different types of assays you wish to perform now and potentially in the future. If in the future you would consider doing fluorescence polarization (FP), select an instrument rated to perform FP and one with a high-quality PMT for maximum sensitivity. If you’re considering doing kinetics assays or ion indicators on live cells, an automated multichannel dispensing is required. 

Some commercially available proteins and enzymes have their storage and stability information clearly defined in the Certificate of Analysis or Product Manual. When this information is provided, follow these directions carefully. 

For protein or enzyme samples that will be derived by the researcher, if this protein/enzyme has been extracted before and information is available in published references, learn everything you can about that protein or enzyme. The first published papers that extracted the protein/enzyme of interest will often include information on its behavior at various temperatures, pH, salt concentrations, buffer systems, and what is required to maintain the activity or stability of the protein/enzyme. Even the same enzyme/protein derived from a different organism within the same class, subclass or order may offer some clues as to its properties. For example, if the enzyme/protein of interest will be extracted from mouse, literature for the same enzyme extracted from rats, humans or other mammals may provide some insight as to its properties. If the enzyme/protein of interest is to be extracted from bacteria, fungi or algae, information on similar enzyme/proteins extracted from mammals or insects may not be pertinent. 

Avoid storing proteins/enzymes in smaller aliquots. At smaller volumes the proteins/enzymes are exposed to greater surface area at the air/liquid interface and with the surface of the container. Such exposure promotes denaturation and/or aggregation. 

When directives are provided on the Certificate of Analysis or Product Manual, such as “Do not aliquot”, do not deviate from these requirements.

This would depend upon either the substrate/reagent or the enzyme/protein of interest. 

Lysed samples can be used if the enzyme/protein of interest is unique and other enzymes/proteins in the lysed sample do not compete for the same substrate/reagent. Lysed samples are not recommended if endogenous enzymes/proteins can compete for the substrate/reagent. For example, the Amplex™ Red kits require the use of an added horseradish peroxidase in converting the dye to resorufin in a stoichiometric reaction with H₂O₂. Endogenous catalases, peroxidases and oxidases can also interact with H₂O₂, limiting detection of actual amount of H₂O₂ generated in the reaction.

Ideally, it is best to work with tissue samples immediately. Although many enzymes/proteins can survive freezing in tissue samples, the time in frozen storage would affect their activity/stability, even if stored in liquid nitrogen. Tissue samples stored frozen over different times cannot be thawed out on the same day and expected to provide reliable results. 

Assuming the enzyme/protein is stable frozen, and even if steps are taken to flash freeze samples, degradation can occur upon thawing. Efforts should be taken to inhibit any biological processes that can degrade the enzyme/protein of interest before the sample is completely thawed, i.e., treating samples with protease inhibitors or other inhibitors to prevent degradation of the enzyme/protein of interest.

For an acceptable inhibition curve, a minimum ten point, 3-fold dilution of the inhibitor or compound should be used. For example, if the initial maximum concentration to be tested is 10 µM, make a dilution series of 10 µM, 3 µM, 1 µM, 0.3 µM, 0.1 µM, etc. 

If enzyme/protein concentration of a sample is unknown, we suggest making a dilution series to test samples at: undiluted, half-dilution, 1/10th dilution and 1/100th dilution in order to determine the best range for the assay. It may be necessary to use a greater dilution than 1:100, but the initial test range would provide some indication of an acceptable dilution range.

In theory, assuming complete linearity of the assay, one may be able to make a standard curve of only two or three points. For example, the Qubit™ fluorometer encodes algorithms for standard curves based on two or three data points for standards provided in the kit. In reality, the more points, the better the fit. A minimum of five-points for a standard curve is acceptable in most cases, but for some assays, there may be lower concentration curve and a higher concentration curve that require a minimum of five points each, as each standard curve may be derived with two different instrument settings of the voltage of the PMT or the gain.

• Conversion of substrates or reagents in the absence of any enzymes/proteins is often due to microbial or biological contamination of materials, surfaces, reagents or even water. If the surfaces or materials are dry and any microbes are dead, their enzymes/proteins may still be active, even in desiccated cells or cellular debris. For example, RNases from sloughed off skin cells are still active. 
• Ultrapure water may be derived for water treatment systems, but the spigot or tubing attached to spigots are surfaces that can become contaminated and in turn contaminate the water as it is dispensed.
• Aggregation or sequestering of either the substrate/reagents, the enzymes/proteins of interest, or any component used in the reaction. All components must be freely soluble, and active sites or binding sites must be accessible. 
• Initial lags or sudden bursts of activity or inconsistent binding during the reaction incubation time may be due to either substrate/reagents initially aggregated gradually or suddenly dissolving or other components aggregating or disaggregating. 
• Assay reagents/materials precipitating out during or after the incubation period for the assay. All components, including the enzymes/proteins, should be soluble and stable for the entire duration of the assay. 
• Materials sticking to the surfaces of pipette tips, vials or cuvettes/microplate wells. Charged surfaces will bind materials of opposite charge. Hydrophobic surfaces will bind hydrophobic molecules. Even rinsing the contents of a pipette tip up by and up and down motion will not dislodge any components that are binding by charge or hydrophobicity. Substrates/reagents sticking to cuvette or microplate surface result in an inaccurate amount of these materials available for the assay. 
• Handling/dispensing errors. Calibrate pipettors often. Test automated dispensing systems before any assay. 
• If working with whole or lysed cell cultures, ensure that the samples are not contaminated with mycoplasma, parasites/symbionts or any viral infections, unless they are part of the experimental sample to be analyzed. 

When performing any microplate-based assay, experimental errors are derived from variations in mixing, dispensing and the materials in the assay. The same errors occur in cuvette-based assays, with the added issue of timing—the time elapsed from readings taken from the first sample to the last. 

Duplicate samples are the minimum. Samples in triplicate provide better data than samples in duplicate and so on. 

Depending on the reagent, absorbance readings are best performed in glass or quartz cuvettes or microplates. Plastic cuvettes and microplates may work just as well but the plastic must not absorb light in the range required for the reading. If using plastics and UV wavelengths, this can be an issue, as many plastics or their components absorb in the UV range. Check with the manufacturer’s specifications as to the usable wavelength range. 

For fluorescence, black-walled plates are recommended to limit crosstalk between wells. Black-bottom or clear-bottom depends upon the optics of the instrument (i.e., Are samples excited and read from the top or excited from the top and read from the bottom?). Cell-based assays should be done in optically clear, flat bottom microplates as cells will settle to the bottom. 

For luminescence (bio- or chemiluminescence), white plates are recommended. 

Both fluorescence and luminescence-based assays may be performed in clear plates, in 96-well or larger well size microplates, but 384-well and smaller well microplates must be either black-walled or white plastic, respectively. 

This depends on the instrument, instrument settings, the detection reagent, the labware and the samples. Adjusting the gain, the voltage of the PMT and light intensity can make the instrument more or less sensitive. Examine unstained, unlabeled samples for color or autofluorescence under wavelengths that will be used in the assay. It may be necessary to subtract out natural color/autofluorescence from experimental samples. For very low concentrations, using less detection reagent may reduce background noise. Eliminate any possible contaminants. In cases where you aren’t given a specific type/brand of microplate or cuvette to use, compare different microplates or cuvettes, to see which ones provide the best signal, the most linear standard curve and the least background. 

All parameters should be the same. Samples should be assayed using the same buffers and reagents from the same kit. Standards from one kit should not be combined with experimental samples using another kit, even if from the same lot number. 

For microplate-based assays, standards and controls should be on the same plate as the experimental samples. If many microplates are to be used, it is not feasible to have a full standard curve and all controls on every plate, you may have a truncated standard curve and the minimum controls on each plate to detect variances in instrument signal due to electrical noise, timing, and other factors. 

This depends upon what is being assayed. 

• If you’re analyzing a new compound, initial testing on a purified enzyme/protein is recommended. 
• If you’re analyzing the presence of an enzyme/protein in natural samples, the assay may either work well with lysed samples or cannot be used on lysed samples at all. 
• When working with live cells, for intracellular enzymes/proteins, the substrate/reagents must either be cell permeable or some delivery mechanism must be employed. Final detection may require the cells to be lysed, but initial application requires that the reagents get into live, intact cells and that the cells do not actively efflux substrate/reagent.

• Start with knowledge of the inherent properties of the protein/enzyme
•  Use only high-quality reagents, buffers, including the quality of the water 
•  Keep everything sterile; use ultrapure water to limit bioburden
•  Avoid making very small aliquots (typically <50 µL)
•  Avoid repeated freezing/thawing and when thawing out, thaw on ice 
•  Do not store proteins/enzymes in frost-free freezers (frost-free freezers have a repetitive heating and cooling cycle to remove frost)
•  Do not store proteins/enzymes in the same refrigerator/freezers that contain volatile organics; trace exposure to organic solvents can cause the denaturation of proteins or inactivate enzymes
• If storing in a freezer that has a lot of use (repeated opening and closing by other users), store the material in a small styrofoam cube with a blue ice block to guard against temperature fluctuations
•  Prior to performing the assay, determine that the protein/enzyme is both soluble and stable under the reaction conditions and for the duration of the assay
•  Where directed, specific conditions for storage and stability posted on Certificates of Analysis or Product Manuals should be followed

Not all proteins/enzymes can be isolated in pure form; they may require accessory proteins or chaperonins for activity or require that hydrophobic domains are covered with lipids, such as with membrane proteins/enzymes. Even proteins/enzymes isolated by affinity purification may co-elute with other cellular components that do not dissociate easily. 

Validity is established by understanding what else is bound to the protein/enzyme of interest and how those other components can affect the assay. Ideally, the material should be analyzed by PAGE and other analytical methods to determine total protein concentration and concentrations of other components (e.g., lipid content, polysaccharide content, metal ion content, etc.).

If your sample is hydrophobic, it may bind to any lipids bound to the protein/enzyme that never enter the active or binding site, the same issue as having the compound bind to the surfaces of microplate wells or cuvettes. A direct assay may help with such samples. 

If the presence or absence of an impurity affects the activity or binding properties of a protein/enzyme, different lots of the protein may vary in the amount/ratios of these impurities may result in variations in the final results. 

GTPases, which act as molecular switches that regulate cell signaling by cycling between GTP-bound (active) and GDP-bound (inactive) state, can be pulled down using an immobilized GTPase-binding domain of downstream proteins that are recruited to GTP-bound, activated GTPases. Read more about GTPase enrichment here.