Product Literature

Catalog: Protein sample prep and quant for MS

Product Literature

Protein sample prep for MS

Manual / Product Insert

Protocol: 1-D PAGE Cleavable ICAT® Reagent Applications Development Kit for Targeted Protein ID and Quantitation (Monoplex Version): P (English )

Version: 05 Sep 2010
Catalog #
  • 4348367(Discontinued)
  • 4339035-LSG
  • 4339036-LSG
  • 4339038-LSG
  • 4339039-LSG
  • 4339040-LSG

Manual / Product Insert

FluoReporter Biotin Quantitation Assay Kit For Biotinylated Proteins

Version: B30757 B30758 06-20-2005

Product FAQ

What are the fluorescent protein assays you offer and how do they differ? Which one should I choose for my samples?

Answer

  • The Quant-iT and Qubit protein assays are easy and accurate assays for the quantitation of protein samples ranging from 12.5 µg⁄mL to 5 mg⁄mL. These assays are highly selective for protein and exhibit very little protein-to-protein variation. Common contaminants, such as salts, solvents, or DNA (but not detergents) are well tolerated in these assays. The Qubit Protein Assay Kit is designed specifically for use with the Qubit Fluorometers, while the Quant-iT Protein Assay is designed to be used both on the Qubit fluorometer or other fluorometers and fluorescence plate readers. 
  • The NanoOrange Protein Quantitation Kit is a very sensitive and easy assay for protein quantitation, with detection as low as 10 ng/mL of protein in solution with a useful assay range of 10 ng/mL to 10 µg/mL. Common contaminants, such as salts, solvents, or DNA (but not detergents) are well tolerated in these assays. This fluorescent dye is suitable for use with spectrofluorometers and microplate readers. 
  • The CBQCA Protein Quantitation Kit is a very sensitive assay for protein quantitation, with detection as low as 10 ng/mL of protein in solution with a useful assay range of 10 ng/mL to 150 µg/mL. CBQCA covalently modifies glutamine, asparagine and primary amines and requires cyanide for the reaction. This assay is tolerant of detergents, but amines, ammonium ions and reducing agents should be avoided. CBQCA is better suited for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides.
  • The EZQ Protein Quantitation Kit provides accurate quantitation of protein samples in the range of 20 µg/mL to 5 mg/mL. The assay can be performed in the presence of detergents, urea, reducing agents, salts, solvents, dyes, and most other contaminants and is generally intended for samples prepared for 1D and 2D gel electrophoresis. 1 µL of each sample is spotted onto filter paper placed inside a specially-designed 96-well microplate, contaminants are washed away, and then the remaining bound proteins are stained with our proprietary fluorescent dye. The paper is then analyzed on a microplate reader or a laser scanner.
  • All the above assay kits come with either concentrated assay reagent and dilution buffer or a pre-diluted quantitation reagent and protein standards. The EZQ Protein Quantitation Kit also comes with a specially-designed 96-well microplate and filter paper that fits inside this microplate.

    Answer Id: E15566

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    Product FAQ

    How can I determine the degree of protein labeling?

    Answer

    To quantitate biotin, we offer two kits as follows:


  • Biotin Quantitation Kit (Cat. No. 28005): With this kit, a solution containing the biotinylated protein is added to a mixture of HABA reagent (4’-hydroxyazobenzene-2-carboxylic acid and avidin. Because of its higher affinity for avidin, biotin displaces the HABA and the absorbance at 500nm decreases proportionally. 
  • Fluorescence Biotin Quantitation kit (Cat. No. 46610): This microplate-based biotin assay is easy to perform by adding the supplied fluorescent reporter to the biotinylated samples and diluted biocytin standards. The avidin fluoresces when the weakly interacting HABA (4’-hydroxyazobenzene-2-carboxylic acid) is displaced by the biotin. The amount of biotin is determined by comparing the sample's fluorescence to the biocytin standard curve. This assay requires must less sample volume than the microplate colorimetric HABA assay and is much more sensitive

  • To determine the dye-to-protein ratio after fluorophore conjugation, absorbance readings of the protein:dye conjugate are taken and the molar ratio can then be calculated. Please go to this Tech Tip (https://tools.thermofisher.com/content/sfs/brochures/TR0031-Calc-FP-ratios.pdf")for more information

    Answer Id: E15705

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    Product FAQ

    I am getting inaccurate results/poor reproducibility with the CBQCA Protein Quantitation Kit. Can you offer some tips?

    Answer

    Here are possible causes and solutions:

    - 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 degress C for several days or at -20 degrees C for long-term storage.
    - 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.
    - pH is too high or too low: Dilute standards and samples in 100 mM sodium borate buffer, pH 9.3. 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.
    - 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.
    - Sample buffer contains high concentrations of thiols: PThiols should not exceed 100µM final concentration in the assay. Add a higher concentration of N-ethylmaleimide (NEM) to block thiols.
    - 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.

    Answer Id: E15594

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    Product FAQ

    What are SureQuant assay kits and what are the different types of SureQuant kits you offer?

    Answer

    SureQuant kits enable multiplex immunoprecipitation to mass spectrometry (mIP-MS) for the simultaneous enrichment and quantitation (absolute or relative) of multiple total and phosphorylated proteins (13 in total, including 11 phosphorylated states) in the AKT/mTOR signaling pathway.

    For each pathway, we offer i) a pathway-specific IP-MS sample preparation module which includes all the reagents necessary to immunoenrich AKT/mTOR pathway proteins and perform in-solution MS sample preparation from sample lysis through peptide clean-up and ii) a quantitation module (relative or absolute), which includes a system suitability standard to monitor MS performance and AQUA Ultimate Heavy and /or Light Peptide mixtures for peptide quantitation. For each pathway, we also offer iii) a multiplex panel which is a combo kit comprising both IP-MS sample preparation and quantitation modules. Two versions of each multiplex panel are offered with either a relative or absolute quantitation module.

    Answer Id: E17266

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    Product FAQ

    I’m seeing other kit-related problems besides the “Standards incorrect” message with my Qubit assay. What do you suggest I try?

    Answer

    Here are several suggestions:

    1.View the raw fluorescence value (RFU) for the standards under “Check Standards” or “Check Calibration”. Confirm that the values for the samples fall between the values of the standards (or a little above the highest standard). If they do not, the sample is out of the accurate range of the assay. Refer to the confidence ranges for each assay in the product manuals. The readout in the assay will be to 2 significant figures instead of 3 if the assay sample is out of the high confidence range.
    To bring the sample into the accurate range, dilute the sample or use more or less of it (for example, 10 µL instead of 2 µL if the sample reads low).

    2.Check for temperature issues: The assay is temperature sensitive and the fluorescent signal can decrease at higher temperatures. Temperature fluctuations between samples, or between samples and standards, can cause problems. Make sure that the buffer and Qubit reagent in DMSO are at room temperature. The buffer and Qubit reagent should be stored at room temperature, not in the refrigerator. Even after 2-3 hours at room temperature, buffer previously stored at 4°C can remain below room temperature. Make sure your samples and working solution are not too warm (including those straight from a centrifuge). Samples kept in the Qubit instrument too long or read multiple times can warm up. If you want to perform multiple readings of a single tube, you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading. Also, do not hold tubes in your hand for very long before reading them in the instrument, since this can warm the sample, resulting in a low reading.

    3.Ensure that you have prepared the Qubit working solution correctly (1:200 dilution using the buffer provided in the kit). Ensure that you have prepared the standard tubes correctly (10 µL of each standard in 190 µL of the working solution). Ensure that the tubes are filled with at least 200 µL (both standards and samples).

    4.Ensure that the reagents and standards you are using are less than 6 months old, and that the standards have been stored correctly. The Qubit reagent stock solution should be protected from light as much as possible.

    5.Ensure that you have selected the correct assay on the Qubit Fluorometer for the Qubit assay you are performing.

    6.Ensure that the lid is completely closed when reading standards and samples.

    7.Use recommended tubes (both so the tube does not obstruct the lid, and for optical clarity). Some types of tubes can have high autofluorescence that will affect the assay.

    8.Did you enter the number of microliters of stock you pipetted into the working solution into the Qubit instrument? If so, the reading after giving the Qubit Fluorometer this information is the concentration of your stock solution. If you did not, the reading you got is for the concentration in the assay tube (the tube you put into the Qubit Fluorometer) and not your stock solution.

    9.If you are comparing Qubit assay results to concentration obtained by UV absorbance, and the concentration based on absorbance is significantly higher, it may be because of nucleic acid or protein contamination. The Qubit assays are much more specific for DNA, RNA, or protein than absorbance readings.

    Answer Id: E8149

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    Product FAQ

    I am getting inaccurate results/poor reproducibility with the Qubit Protein Assay Kit. Can you offer some tips?

    Answer

    Here are possible causes and solutions:

    - 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) or Axygen PCR-05-C tubes. 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: 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 (https://tools.thermofisher.com/content/sfs/manuals/Qubit_Protein_Assay_UG.pdf).
    - 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 (https://tools.thermofisher.com/content/sfs/manuals/Qubit_Protein_Assay_UG.pdf) 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.

    Answer Id: E15593

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    Manual / Product Insert

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