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We are dedicated to your success. Get back on track. View our expert recommendations for commonly encountered problem scenarios related to your protein assays and quantitation experiments.
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Each method has its particular sensitivities. Apart from the information presented in the product specific instruction booklet, we offer an expanded protein assay compatibility table in this Tech Tip.
Often, an alternative wavelength can be used, although the slope of the standard curve and the overall assay sensitivity will be reduced. Our Tech Tip offers additional information on determining acceptable wavelengths for measuring protein assays.
The sample likely contains an incompatible substance. Refer to the information in the product specific protocol or our Tech Tip.
Several strategies exist for overcoming or eliminating sample incompatibility with protein assays. The simplest method is to assay the sample after diluting it several-fold in a compatible buffer. If the starting concentration of protein is sufficient to remain within the protein assay working range upon its dilution, then this method will often reduce the amount of interfering substance in the sample to the point where it no longer interferes. Another method is to dialyze or desalt samples into a buffer that is compatible with the assay.
Precipitation can be used to eliminate interfering substances. After causing the protein to precipitate with acetone or trichloroacetic acid (TCA), the supernatant containing the interfering substance can be removed. Then the protein pellet is dissolved in the assay working reagent, and the protein assayed performed as usual. A general protocol for protein precipitation is provided in our Tech Tip.
Each protein assay method has its particular sensitivities. Generally, the following substances should be avoided for the given protein assay method:
It is possible to have a substance additive affect such that even though a single component is present at a concentration below its listed compatibility, a sample buffer containing a combination of substances could interfere with the assay. You should take steps to eliminate or minimize the effects of the interfering substance(s) by diluting or removing the substance.
You can test the tolerance of the assay for your specific buffer formulation. For in-house generated compatibility information, substances were considered compatible at the indicated concentration in the Standard Test Tube Protocol (found in the manual for each protein assay) if the error in protein concentration estimation caused by the presence of the substance was less than or equal to 10%. The substances were tested using WR prepared immediately before each experiment. Blank-corrected 562nm absorbance measurements (for a 1000µg/mL BSA standard + substance) were compared to the net 562nm measurements of the same standard prepared in 0.9% saline.
Each of the commonly used total protein assay methods exhibits some degree of varying response toward different proteins. These differences relate to amino acid sequence, pI, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein’s color response. Most protein assay methods use BSA or immunoglobulin (IgG) as the standard against which the concentration of protein in the sample is determined. However, if great accuracy is required, prepare the standard curve from a pure sample of the target protein.
There are two potential causes for low absorbance values in the unknown samples. The first being that pH and other buffer conditions were not optimal for the protease being tested. You can repeat assay using buffer conditions that are optimal for the protease being tested and used to make standard curve. Secondly, it is possible that the protease being tested digests the casein substrate more slowly or less fully than the trypsin standard for which the default assay method was developed. You can perform the digestion step for >20 minutes (e.g., 40 minutes); if necessary, also perform the TNBSA development step for longer. You can also use the specific substrate for the protease being tested.
It could be that the sample protease activity is low relative to the range of dilutions used for the standard curve. You can prepare and use additional standard dilutions to ensure that the full range of assay sensitivity is utilized. Alternatively, the sample protease activity is low relative to the protease used to make standard curve. You can prepare a standard curve using the known amounts of the specific protease being tested.
Check the scale used for your X-axis. A logarithmic scale should be used for the X-axis (protease enzyme concentration) rather than a linear scale.
Ensure that the instrument gain setting is sufficiently low to avoid saturating the instrument detector. For fluorescence readers containing multiple excitation/emission positions, ensure that “top/top” is selected for excitation/emission settings.
Variations in pipetting can directly contribute to assay error, especially in FRET-mode, because the working reagent itself has some intrinsic background fluorescence. Use reverse-pipetting or other technique to prevent the introduction of small air bubbles into the plate wells. In FP-mode, if the sample matrix contains very high amounts of interfering fluorescent material, better quality results may be obtained by subtraction of buffer blanks from the data used for mP calculation. In addition, FP detection is very sensitive to the position of the optical head in relation to the center of the well with respect to its X and Y coordinates, especially with 384-well plates. This position-artifact may be identified as reproducible patterned data by positioning the plate for a second reading 180 degrees relative to the first reading. Ensure that the instrument read positioning settings are appropriate for the type of plate chosen.
No. The filter paper that is supplied with the EZQ™ Protein Quantitation Kit is a very specific filter paper and the identity is proprietary. Most lab filter papers will not provide similar results as the paper provided in the kit, so substitutions are not recommended.
Yes, once the samples have dried onto the filter paper, the filter paper can be stored and stained at a later date. After the staining procedure is complete, the signal is very stable, so the paper can be dried again and scanned at a later date as well. The paper can be scanned dry or after dipping in water. If storing the paper before staining, we recommend storing in a plastic bag to prevent contamination that would affect the staining pattern.
Protein on the skin will transfer to the filter paper and be stained with the EZQ™ Protein QuantitationReagent. We recommend handling the filter paper with tweezers and cleaning the staining dish and tweezers before use to minimize marks.
It is not unusual for the filter paper to bend and warp on drying. It is very important that the paper be flat when scanning or the signal will be uneven and give very inaccurate quantitation values. If this is a problem then wet the paper in water and scan wet.
We have changed the manufacturer of the Qubit™ Assay Tubes and the new tubes are slightly longer and more opaque than the original tubes. The new tubes have been tested in each of the Qubit™ instruments, with all of the Qubit™ assays, and using the fluorescent dyes, fluorescein and Alexa Fluor™ 647, to validate that there are no changes to the data quality when compared with the original Qubit™ tubes. The new tubes met or exceeded the current specifications for both the core and extended ranges for each of the Qubit™ assays, and were able to achieve the same limit of detection (LOD) as the original Qubit™ tubes for each of the Qubit™ assays and the fluorescent dyes tested.
Here are possible causes and solutions:
| Cause | Solution |
The kit has expired or has been stored incorrectly | When properly stored, the components of the Qubit™ Protein Assay Kit should be stable for at least 6 months. Upon receipt, the kit can be stored at 4 degrees C. Components A and B can be stored at ambient temperature and Components C-E can be stored at ≤4 degrees C. Protect Component A from light. |
| High degradation of the BSA standard 2 and 3 will result in a decrease in signal and a “Standards Incorrect” error warning upon calibration. Replace the kit. | |
Old calibration data was used | Best practice is to prepare fresh calibration standards at the same time as the samples to take into account any changes in assay conditions. |
Incorrect tubes were used | Use the recommended Qubit™ Assay Tubes (Cat. No, Q32856). Other 0.5 mL thin-walled PCR tubes may work as well, but performance is not guaranteed. Avoid opaque tubes, as these will block the light path. |
Tubes contain bubbles or particulates that are scattering the light | Pipette gently to avoid the introduction of bubbles. Spin down tubes before measuring to remove bubbles or particulates. Spin down samples to remove particulates before adding an aliquot to the Qubit™ working solution. |
Inaccurate pipetting | The Qubit™ assay will accept 1-20 µL of sample, but pipetting very low volumes, especially 1-2 µL is typically very inaccurate, especially with viscous samples. If possible, pipette at least 5 µL for more consistent results. |
The temperature of the assay is changing | Make sure that the Component B buffer is at ambient temperature before use and avoid leaving the samples in the Qubit™ instrument or near an exhaust fan or other heat source that would warm up the samples. |
Contamination in buffer causing high background | High buffer contamination will show up as an increase in the relative fluorescence (RFU) of the background, measured with the standard tube 1 blank, and eventually will trigger a “Standards Incorrect” error warning on calibration. Replace the kit. |
Sample buffer contains detergents | The Qubit™ protein assay is a detergent-based assay, utilizing an environmentally sensitive dye that fluoresces in the presence of detergents; therefore, only very low concentrations of additional detergents are tolerated in the assay, as listed in Table 2 in the manual. |
Sample buffer contains other components that are affecting the assay | The Qubit™ protein assay is generally tolerant of reducing reagents, salts, free nucleotides, amino acids, DNA, and solvents. Table 2 in the manual lists acceptable concentrations for many common contaminants. If you have a contaminant you think is affecting the quantitation, then prepare duplicate standard tubes, and spike the contaminant into one set of tubes and run them as samples. If the effect is not too substantial, then spike the buffer into the standards when performing the calibration to account for buffer composition effects. |
Here are possible causes and solutions:
Cause
| Solution |
The kit has expired or has been stored incorrectly | When properly stored, the components of the CBQCA Protein Quantitation Kit should be stable for at least 6 months. Upon receipt, the kit should be stored at -20 degrees C and protected from light. Solutions of Components A, C, and D may be stored at 4 degrees C for several days or at -20 degrees C for long-term storage.
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Poor excitation/emission filter settings | Read the samples in a fluorescence microplate reader, standard fluorometer or minifluorometer using excitation/emission wavelengths of approximately 465/550 nm.
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pH is too high or too low | Dilute standards and samples in 100 mM sodium borate pH 9.3 buffer. Other buffers can be used, but labeling is optimal near pH 9.3. Prepare standards and samples under the same conditions to account for buffer pH effects.
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Sample buffer contains primary amines or ammonium salts | The CBQCA reagent reacts with glutamine, asparagine and primary amines, such as the epsilon amine on lysine. It does not react with histidine or secondary amines. Therefore the sample should be free of ammonium salts and contaminating amines such as Tris or glycine.
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Sample buffer contains high concentrations of thiols | Thiols should not exceed 100 µM final concentration in the assay. Add a higher concentration of N-ethylmaleimide (NEM) to block thiols.
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Sample buffer contains other components that are affecting the assay | The CBQCA assay is generally tolerant of the presence of lipids, detergents, glycerol, sucrose, salts and sodium azide, but these contaminants do affect the signal and sensitivity. Prepare standards and samples under the same conditions to account for buffer composition effects.
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