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The A450 values that I got for the standard curve in your ELISA kit are lower than the example values shown in the product manual. Why? Product FAQ

Answer

There are 2 main causes of poor ELISA standard curves. First, the recommended method for solubilizing the kit standard may not have been followed. The standard should be reconstituted according to the directions indicated on the label, using the standard diluent provided in the kit. No other diluent should be used. The vial should then be swirled or mixed gently and then allowed to sit for 10 minutes at room temperature to ensure complete solubilization. This concentrated standard solution should be used within 1 hour of reconstitution. Also, it should be mixed gently again before preparing the dilutions in standard diluent according to the instructions provided in the product manual. Leftover standard can usually be stored frozen in small aliquots, unless specified otherwise in the product manual.

The second common reason for poor standard curves is that the HRP conjugate was not diluted correctly. The 100X HRP conjugate solution contains 50% glycerol, which makes it very viscous and difficult to pipet accurately. Here is what we suggest to solve this problem: First, let the vial of HRP conjugate come to room temperature. Then, stir it gently with a clean pipet tip to make sure that it is homogeneous. Use only the separate HRP conjugate diluent provided in the kit to dilute it, and follow the dilution instructions provided in the manual.

The key to diluting the HRP conjugate is to make sure that it is pipetted correctly. You should test that your pipettor accurately aspirates and dispenses the volume of the conjugate-glycerol mixture that is required. If possible, this pipettor should be calibrated so it is accurate and reliable. When you aspirate the viscous conjugate solution, it may take 5-10 seconds for the desired amount to enter the pipet tip. Before transferring the conjugate to the appropriate HRP diluent, make sure that the outside of the pipet tip is dry by wiping it with a lab tissue (e.g., Kimwipes tissue), taking care to ensure that the contents inside the tip do not get absorbed by the tissue. Pipet the conjugate into the diluent, and then rinse out the tip by pipetting up and down several times. It is important to get every last bit of conjugate out of the tip. Next, seal the container and mix it gently but thoroughly by rocking it or turning it upside down. This is crucial because the glycerol carries the conjugate quickly down to the bottom of the tube. If the diluted conjugate is not mixed adequately, the concentration of the HRP conjugate will not be what is required.

Once the HRP conjugate is diluted and mixed gently but well, use it within 15 minutes. Remember that the HRP conjugate diluent is the only acceptable diluent for the HRP conjugate. The diluted HRP conjugate should not be saved because the HRP activity is labile, and it should never be stored and reused.

Answer Id:: E12628

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What is the difference between the Cytoscreen ELISA format and the EASIA format? Product FAQ

Answer

Our EASIA (Enzyme Amplified-Sensitivity Immuno Assay) kits are three-member solid-phase sandwich assays, also called direct ELISA kits. They contain 96-well plates coated with one or more F(ab')2 monoclonal capture antibodies. The standards in these kits are provided as lyophilized powders containing analyte at discrete levels plus plasma proteins. Standards are reconstituted by adding water. EASIA kits also include lyophilized controls with known levels of analyte in plasma protein. In performing the EASIA, sample, standard, or control is pipetted onto the plate. Next, horseradish peroxidase (HRP) conjugated detection antibody is added to complete a three-member solid-phase sandwich. Following a washing step, the HRP activity is quantitated by incubation with substrate solution (TMB plus H2O2). At the end of the incubation step, the HRP reaction is terminated by the addition of an acidic stop solution and the optical density of the solution in the wells is measured with a spectrophotometric ELISA plate reader. EASIA kits are validated for measuring human serum, plasma, culture supernatant, and other biological fluids.

Our Cytoscreen ELISA kits also contain pre-coated 96-well plates. The standard curve is constructed by reconstitution and serial dilution of a single vial of standard using standard diluent buffer provided in the kit. After samples or standards are pipetted into the wells, a biotin-labeled detection antibody is added, followed by a wash step and addition of HRP-conjugated streptavidin to complete a four-member solid-phase sandwich. As with the EASIA kits, the HRP activity is quantitated by incubation with substrate solution (TMB plus H2O2) followed by an acidic stop solution. The optical density is measured with a spectrophotometric ELISA plate reader. Cytoscreen ELISA kits are validated for measuring analytes in serum or plasma, culture supernatant, and other buffered solutions, and are available for quantitation of human, mouse, rat, monkey, and pig samples.

Answer Id:: E5149

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I have bought several ProcartaPlex Simplex Bead Sets. Which premixed standard do I have to use in my assay? Product FAQ

Answer

Please refer to the lot-specific Certificate of Analysis to check the source of the Standard Mix (i.e., Standard Mix A, B, or C) included with each kit. When combining Simplex Bead Sets with other Simplex Bead Sets or Multiplex kits, multiple vials of the same premixed standard (Standard Mix A, B, or C) may be shipped. If the combination of analytes you want to analyze in your multiplex assay includes two analytes that require the same premixed standard, use only one vial of the premixed standards to prepare the standard curve. If the kits you want to combine include different lots of the same premixed standard, please choose one vial (of any lot) for your multiplex assay.

Answer Id:: E14587

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Can I combine Multiplex Luminex assays for higher-level multiplexing? Product FAQ

Answer

While most of our Multiplex Luminex Assay kits can be combined with other Singleplex Assay kits to permit higher-level multiplexing, there are a few exceptions and limitations. Please carefully read the product manual for kit-specific guidelines. Instructions for combining bead mixtures and Detector Antibody mixtures in the development of multiplexed assays are shown below.

Note: Before preparing multiplexed assays, it is important to verify that each analyte is represented by a unique bead region. This assures the compatibility of each bead in the development of multiplexed assays. Up to 10 bead concentrates (premixed panel multiplexes and/or singleplexes) can be combined to increase the number of proteins being monitored. The buffer systems used for each kit must also be compatible. In general, Luminex assay kits that use Buffer Kit Cat. No. LHB0001 should not be multiplexed with Luminex Assay kits that use Buffer Kit Cat. No. LHB0002. Also, protein standards may be analyzed alone or combined with other protein standards for higher levels of multiplexing. Do not combine more than 4 vials.

Multiplexing Standards:
Protein standards may be analyzed alone, or combined with other protein standards for higher levels of multiplexing. Do not combine more than 4 vials.
One vial standard: Reconstitute the standard vial in the suggested reconstitution volume, ususally 1 mL, of appropriate diluent.
Two vials of standards: Reconstitute each vial with 0.5 mL of appropriate diluent. Combine 300 µL from each vial and mix by gently pipetting up and down 5 to 10 times.
Three vials of standards: Reconsitute each vial with 0.333 mL of appropriate diluent. Combine 200 µL from each vial and mix by gently pipetting up and down 5 to 10 times.
Four vials of standards: Reconsitute each vial with 0.250 mL of appropriate diluent.

Answer Id:: E12652

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I am getting an elevated background in my ELISA. What should I do? Product FAQ

Answer

Here are possible causes and solutions:

Insufficient washing and/or draining of wells after washing. Residual solution containing anti-rabbit IgG HRP or streptavidin-HRP can elevate the background if left in the well.
Wash according to the protocol. Verify the function of the automated plate washer. At the end of each washing step, invert the plate on absorbent tissue on the countertop and allow it to completely drain, tapping forcefully if necessary to remove residual fluid.
Chromogen exposed to light prior to use, resulting in a blue color.
Keep chromogen in its vial until you are ready to dispense it into the plate, and then pour it into a reservoir to prevent contamination of the vial with equipment. Do not cover the plate with foil.
Incubation time is too long or incubation temperature is too high.
Reduce incubation time and/or temperature.
Contamination of pipette, dispensing reservoir, or substrate solution with anti-rabbit IgG HRP or streptavidin-HRP.
Do not use chromogen that appears blue prior to dispensing onto the plate. Obtain a new vial of chromogen.
Blanks that have been set up improperly.
Follow the protocol when designating blank wells. Blank wells contain only chromogen and stop solution. Subtract blank well results from all other wells.
Incorrect dilution of standard stock solution or standards diluted in serum, culture supernatant, or other.
Follow the protocol instructions regarding dilution of the standard. Dilute standards only in the Standard Diluent Buffer provided in the kit.

Answer Id:: E12630

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I am getting lower protein yield than expected with the ExpiSf Expression System. What should I do to improve my results? Product FAQ

Answer

Protein yield can vary greatly from protein to protein. We strongly recommend using the pFastBac 1 - Gus positive control vector (included in the ExpiSf Expression System Starter Kit as well as sold separately - Cat. No. 10360014) to generate Gus-expressing baculovirus and express Gus recombinant protein. The typical yield of Gus protein using the ExpiSf Expression System is approximately 250,000 activity units/mL following the recommended quantification protocol described in the ExpiSf Expression System User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017532_ExpiSfExpressionSystem_UG.pdf). Quantification of the Gus positive control protein will help determine if the low protein yield is due to a low expressing protein, low baculovirus titer/MOI used, poor baculovirus stock quality, a problem with the system components, or transfection and cell culturing conditions. If the expected protein yield is not being achieved with the Gus positive control, we recommend checking the following:

1. Ensure that you have a healthy ExpiSf9 cell culture:
- Cells are >90% viable during normal passaging and at the time of transfection and infection.
- Cells exhibit a doubling time of approximately 24 hrs.
- Cells recover rapidly post-thaw (i.e., reach a cell density of 5 x 10E6 cells/mL with ≥80% viability within 4-5 days post thaw); if not, verify that the culturing guidelines provided in the product manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017532_ExpiSfExpressionSystem_UG.pdf) are being followed, and thaw a new vial of cells if necessary.
- Verify that the incubator temperature is set at 27 degrees C for your cultures as temperatures higher than 28 degrees C may cause cells to overgrow and display reduced yield capacity overtime. Verify that the shake speed is 125±5 rpm for shakers with a 19 mm or 25 mm orbital diameter and 95±5 rpm for shakers with a 50 mm orbital diameter. All of our shake speed recommendations are provided for Nalgene PETG non-baffled Erlenmeyer flasks; if a different culture vessel is being used, additional shake speed and/or other culture parameter(s) optimization may be required.
2. Ensure that you have a high-quality baculovirus stock:
a. Ensure that pure and high-quality bacmid DNA is used:
- Check the quality of the recombinant bacmid by agarose gel electrophoresis to confirm DNA integrity.
- Ensure that a single white colony is picked during bacmid preparation to avoid a mixture of recombinant and non-recombinant baculovirus.
- Confirm that pure bacmid DNA is used (i.e., contains only recombinant bacmid and no empty bacmid), consider screening other DH10Bac transformants (i.e., white colonies).
- Look for the typical signs of a good transfection reaction: When ExpiSf9 Cells are efficiently transfected and baculovirus is being produced, viable cell density is between 80-90% at Day 3 post-transfection and will decrease to <80% at Day 4 post-transfection.
b. Ensure that the recombinant baculovirus contains the gene of interest:
- Verify transposition of bacmid DNA by PCR analysis using the pUC/M13 Forward and Reverse primers.
- Re-transfect ExpiSf9 Cells with new recombinant bacmid DNA, if necessary.
c. We recommend using the suspension-based transfection protocol for generation of high-quality P0 virus, as multiple rounds of viral amplification following the classical adherent format can result in spontaneous excision of the gene of interest as well as the formation of defective viral particles.
d. Ensure that the baculovirus stock is stored properly: Baculovirus stock should be stored at 4 degrees C, protected from light for up to 12 weeks. Alternatively, baculovirus stocks can be frozen and stored at -80 degrees C or in liquid nitrogen (no DMSO or cryopreservative) for longer periods. We do not recommend repeated freeze/thaw cycles of your virus stock. Frozen virus should be stored in small aliquots and not re-frozen once thawed.
3. Ensure that the cells are seeded at 5 x 10E6 viable cells/mL at the time of ExpiSf Enhancer addition.
4. Ensure that the correct amount of ExpiSf Enhancer is added 18-24 hrs prior to infection. Incubating enhancer-treated cells for longer than 24 hrs may result in decreased infection efficiency and low protein yields.
5. Ensure that the cell density at the time of virus infection is at 5-7 x 10E6 viable cells/mL. Infecting cells at higher densities will lead to sub-optimal infection conditions and poor protein yields.
6. Ensure that the optimal volume of baculovirus stock is used to infect cells. We recommend starting with an MOI of 5 and further optimizing the MOI (from 3-10), if necessary.
7. If baculovirus titer wasn't determined, we recommend testing a series of virus stock dilutions to determine the optimal volume to use for virus infections. A starting dilution range of 1:50 - 1:100 (virus stock : culture volume) is a good starting point.

Answer Id:: E16491

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I am interested in generating cDNA from total RNA. What is the difference between SuperScript III Reverse Transcriptase and SuperScript III First Strand Synthesis System for RT-PCR? Product FAQ

Answer

SuperScript III Reverse Transcriptase (Cat. Nos. 18080093, 18080044, 18080085) contains the stand-alone enzyme and a vial each of 5X first-strand buffer and 100 mM DTT.

SuperScript III First Strand Synthesis System for RT-PCR is a complete kit that provides the SuperScript III Reverse Transcriptase and all the other components required for synthesis of first-strand cDNA from total or poly(A)- RNA. It includes:
- Superscript III Reverse Transcriptase
- Oligo (dT)20 Primer
- Random hexamers
- 10X RT buffer
- 25 mM MgCl2
- 0.1 M DTT
- 10 mM dNTP Mix
- RNAseOUT Recombinant Ribonuclease Inhibitor
- E. coli RNAse H
- DEPC-treated water
- Total HeLa RNA control
- Sense control primer
- Anti-sense control primer
Note: The kit does not include the PCR amplification enzyme.

Answer Id:: E7627

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Is it possible to determine cytokine concentration in samples with low protein concentrations, such as cerebral spinal fluid (CSF)? Product FAQ

Answer

To assay cytokines in samples with low protein concentrations with our EASIA (direct ELISA) kits, one thing to consider is the effect of “matrix”. Matrix is the liquid environment which surrounds the cytokine. The composition of the matrix can influence the results of the ELISA. The measurement of cytokines with low protein concentrations such as CSF, tissue culture supernatant, broncheolar alveolar lavage fluid, and urine, may produce results which are not truly comparable with a standard curve generated with serum as the matrix. These matrices with low protein concentration should be modified so that they more closely resemble the matrix used to generate the standard curve. We have found that addition of just a single protein component to samples with low protein matrices, such as adding bovine serum albumin, is not sufficient to cause the matrix to resemble that of the standard curve. We suggest several methods to adjust the matrix of samples so that it more closely resembles that of the standard curve. The first method is to dilute the sample 1:4 (one part sample plus three parts diluent) with the diluent provided in the kit (usually Standard Zero). Alternatively, a pool of serum from healthy volunteers may be used in place of the diluent. If pooled serum is used, it is important that the concentration of the cytokine to be measured first be quantitated in this serum and the appropriate correction should be made. An example is given below:

If the pooled serum is found to have 10 pg/mL of the cytokine of interest and cytokine level in the sample diluted 1 part sample in 3 parts pooled serum is measured to be 50 pg/mL, the calculated concentration in the sample is 50 - (10 x 3/4) = 42.5 pg/mL, then correcting for the 1:4 dilution, 42.5 pg/mL x 4 = 170 pg/mL.

A second method for increasing the matrix protein in samples is designated the Point-0-Cytokine (or Point-0-Receptor, depending on the kit) method. This method should be considered for use with samples that have low cytokine concentrations in a matrix with a low protein concentration. The presence of the cytokine at low concentrations does not permit the researcher to dilute the sample 1:4 because the cytokine concentration in the diluted sample would be below the level of detectability by the assay. The use of the Point-0-Cytokine vials allows the researcher to increase the concentration of the protein without diluting the samples. Each vial of Point-0-Cytokine contains lyophilized serum protein. To use the Point-0-Cytokine vials, the researcher simply adds 500 microliters of the low protein sample to the contents of the vials, and allows the serum proteins to rehydrate. Many of the EASIA kits require Point-0-Cytokine vials for low protein, low cytokine containing samples. Other kits require Point-0-Receptor vials which are used in the same manner as the Point-0-Cytokine. CSF requires addition of PT0 cytokine or PT0 receptor, or the normal procedure can be followed, depending on the kit.

Answer Id:: E5142

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Is there a convenient protocol for multiplexing Luminex assays? Product FAQ

Answer

Yes. We have included the protocol for multiplexing our Luminex Assay kits in the Appendix for the Luminex buffer kit manuals. Also in the Preparing Reagents section of each Luminex buffer kit manual, instructions are provided for multiplexing standards. General instructions are provided below. While most Luminex Assay kits can be multiplexed, there are a few exceptions and limitations. Please carefully read your product manuals for kit-specific guidelines.

Higher Multiplexing:
Before preparing multiplexed assays, it is important to verify that each analyte is represented by a unique bead region. This assures the compatibility of each bead in the development of multiplexed assays. Up to 10 bead concentrates (pre-mixed panel multiplexes and/or singleplexes) can be combined to increase the number of proteins being monitored. The buffer systems used for each kit must also be compatible. In general,Luminex Assay kits that use buffer kit LHB0001 should not be multiplexed with Luminex Assay kits that use buffer kit LHB0002. Also, protein standards may be analyzed alone, or combined with other protein standards for higher levels of multiplexing. Do not combine more than 4 vials.

Multiplexing Standards:
One vial of standard
Reconstitute the standard vial in the suggested reconstitution volume, usually 1 mL, of appropriate diluent.
Two vials of standards
Reconstitute each vial with 0.5 mL of appropriate diluent. Combine 300 µL from each vial and mix by gently pipetting up and down 5 to 10 times.
Three vials of standards
Reconstitute each vial with 0.333 mL of appropriate diluent. Combine 200 µL from each vial and mix by gently pipetting up and down 5 to 10 times.
Four vials of standards
Reconstitute each vial with 0.250 mL of appropriate diluent. Combine 150 µL from each vial and mix by gently pipetting up and down 5 to 10 times.

Multiplexing Antibody Beads:
The volume required for a multiplexed assay can be calculated by using the formulas presented below.
Total Volume of 1X Antibody Bead Mixture Required:
0.0275 mL x (# of wells) = ________________

Volume of each 10X Antibody Bead Concentrate Required:
Total Volume of 1X Antibody Bead Mixture Required = ______ / 11

Working Wash Solution Required:
[Total Volume of 1X Bead Mixture] - [(Volume 10X Beads) x (# of plexes)] =________

To prepare a 1X Antibody Bead mixture for a multiplexed assay, pipette the beads and Working Wash Solution (using the volumes calculated with the formulas presented above) into a foil wrapped tube. Vortex the tube for 30 seconds, then sonicate for 30 seconds. The mixture is ready to be used in a multiplexed assay. If desired, premixed beads can be stored overnight at 2 to 8 degrees C.

Multiplexing Detector Antibodies:
The volume required for a multiplexed assay can be calculated by using the formulas presented below.
Total Volume of 1X Detector Antibody Mixture Required
0.110 mL x (# of wells) = ________________

Volume of each 10X Detector Antibody Concentrate Required
Total Volume of 1X Detector Antibody Mixture Required = ______ / 11

Detector Antibody Diluent Solution Required:
[Total Volume of 1X Detector Antibody Mixture] – [(Volume10X Detector Antibody) x (# of plexes)] =________

To prepare a Detector Antibody mixture for a multiplexed assay, pipette the Detector Antibody Concentrates and Detector Antibody Diluent (using the volumes calculated with the formulas presented above) into a tube. Mix gently. The mixture is ready to be used in a multiplexed assay. If desired, premixed 1X Detector Antibody can be stored overnight at 2 to 8 degrees C.

Answer Id:: E5167

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How do I oxidize a glycoprotein or antibody when using the GlycoLink Immobilization Kit? Product FAQ

Answer

The antibody or other glycoprotein is first diluted or desalted into the coupling buffer and transferred to a vial containing sodium meta-periodate at a final concentration of 10 mM (1 mM is sufficient for sialic acid residues). The mixture is incubated for 30 minutes at room temperature in the dark. Sodium meta-periodate concentration can be increased to 25mM if oxidation is not sufficient at 10 mM. Care must be taken not to over-oxidize the glycoprotein.

Answer Id:: E8227

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Rat IL-1 Ra ELISA Kit Product Information Sheet Manual / Product Insert

Catalog # ERA22RB

Process water products catalog Product Literature

Human TNFRSF18 (lysateS) ELISA Kit Manual / Product Insert

  • Version: Rev 3.0 (25Mar16)
Catalog # EHTNFRSF18CL
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