What is the difference between your total and phosphospecific ELISA kits?

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Both types of ELISA kits capture total protein, regardless of its phosphorylation state, within the wells of a plastic 96-well plate. This is done by coating the wells with a “pan-antibody” that does not distinguish between the phosphorylated and non-phosphorylated forms of a protein and does not block the phosphorylation site to be studied. In addition, a phosphospecific ELISA kit quantifies the amount of that same protein that is phosphorylated on one or more specific amino acids. Instead of a second pan-antibody for detection, this assay uses an antibody that specifically recognizes an epitope that is only present on a protein when it is phosphorylated specifically (i.e., it is phosphospecific).

We recommend running the total and phosphospecific ELISAs simultaneously with the same samples. If this is not possible, make sure to test the same samples with both kits as soon as possible.

Answer Id: E12621

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What units of measurement do you use for the results of total and phosphospecific ELISAs?

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The results of our total ELISAs are given in pg/mL of sample, or sometimes ng/mL. This measurement is always given in mass units because standards of known mass are used to prepare the standard curve. The results of the phosphospecific ELISAs are given in “units”, which we do not relate to a particular mass of protein. We use units because it is difficult to precisely know the efficiency of a particular phosphorylation reaction, and therefore the ratio of phosphorylated to unphosphorylated protein, in a particular preparation of phosphoprotein standard. Phosphorylation units will be unique to each phosphospecific ELISA and are described within the product manual that accompanies each kit.

For example, a typical unit description would be “1 unit = the amount of FAK [pY397] derived from 300 pg of auto-phosphorylated FAK protein”. Since there is no guarantee that the FAK in our standard preparation is 100% phosphorylated, we refrain from making the statement that this corresponds to 300 pg of phosphorylated FAK. Instead, we validate a large batch of phosphorylated protein and use this to develop our unit definition and standard curve for our original assay. Subsequent preparations of our protein standards are normalized to the original batch of protein to ensure that our unit definitions remain constant from lot to lot.

Answer Id: E12622

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How do I normalize phosphospecific ELISA results with total ELISA results?

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In order to evaluate phosphorylation levels, we report comparative levels of protein phosphorylation in units of phosphoprotein per pg or ng of total protein. The total ELISA kit quantifies the mass of protein per sample, and the phosphospecific ELISA kit quantifies the phosphorylation level of that protein in units. One can then determine if phosphorylation levels (in units/pg, for instance) of various samples are similar or different.

Example: Two samples are tested for total CREB and CREB [pS133]
Sample 1 results: The total assay (KHO0231) shows 100 pg/mL of CREB in the sample. The phosphospecific ELISA (KHO0241) shows 50 units/mL of CREB [pS133]. In this sample, CREB is phosphorylated at serine 133 to the level of (50 units/mL)/(100 pg/mL) = 0.5 units/pg of total CREB.
Sample 2 results: The total assay shows 95 pg/mL of CREB in the sample. The phosphospecific ELISA results show 5 units/mL of CREB [pS133]. In this sample, CREB is phosphorylated at serine 133 to the level of (5 units/mL)/(95 pg/mL) = 0.053 units/pg of total CREB. When you compare sample 1 with sample 2, you see a 10-fold difference in the level of phosphorylation of CREB at serine 133, even though the amount of total CREB protein is nearly unchanged.

Answer Id: E12623

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How do I process plasma samples for use on the Luminex™ assay platform?

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Separate the cells from the plasma samples by centrifugation at 2,000 x g for 10 min in a refrigerated centrifuge. Centrifugation at this force is necessary to deplete the sample of platelets. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2-8 degrees C while handling.

If the plasma is to be analyzed at a later date, apportion it into aliquots in polypropylene microcentrifuge tubes and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When you are ready to analyze them, allow the samples to thaw on ice. All plasma samples should be clarified by centrifugation (14,000 rpm for 10 min at 4 degrees C) in a refrigerated microcentrifuge immediately prior to analysis. Follow the assay procedure provided with the kit for appropriate dilutions.

Answer Id: E12641

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How do your phosphospecific ELISA kits compare to immunoprecipitation and western blotting?

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Our phosphospecific ELISA kits have several advantages, including ease of use and increased sensitivity. Phosphospecific ELISA kits are typically 2-10 times more sensitive than western blots, so they are particularly useful for the detection of “low-expressing” proteins or for small sample sizes. In addition, with the use of the recombinant standards provided in the kit, phosphospecific ELISAs provide quantitative results without having to perform densitometry.

Answer Id: E12624

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What is the Luminex™ assay platform?

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Beads of defined spectral properties are conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration, control samples, and test samples) into the wells of a microplate. The target protein binds to the capture antibodies over the course of a 2 hr incubation. After washing the beads, protein-specific biotinylated detector antibodies are added and incubated with the beads for 1 hr. Next, excess biotinylated detector antibodies are removed, and a streptavidin-conjugated fluorescent protein, R-phycoerythrin (SAV-RPE), is added and incubated for 30 min. SAV-RPE binds to the biotinylated detector antibodies, forming a four-member, solid-phase sandwich. After washing to remove unbound SAV-RPE, the beads are analyzed with a Luminex™ detection system. By monitoring the spectral properties of the beads and the amount of associated R-phycoerythrin (RPE) fluorescence, the concentration of one or more proteins can be determined. The Luminex™ technology is compatible with the following Luminex™ analyzers:

MAGPIX™ System-affordable, efficient, and compact
Luminex™ 100/200™ System-versatile, efficient, and widely used in multiplexing
FLEXMAP 3D™ System-high throughput (up to 500 simultaneous assays) and automation compatible

Answer Id: E12635

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How do I process synovial fluid on the Luminex™ assay platform?

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Collect synovial fluid into non-heparinized tubes and spin at 1,000 x g for 10 min within 30 min of sample collection. The acellular portion of synovial fluid should be stored at -80 degrees C before subsequent analysis. All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute samples 1:1 with Assay Diluent prior to addition to the assay. Reference: Raza K et al. (2005) Arthritis Research &Therapy 7(4): R784-R795.

Answer Id: E12645

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How do I process cell culture supernatants for use on the Luminex™ assay platform?

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Cells should be in log-phase growth. Stimulate cells as desired in appropriate cell culture flasks. Using sterile technique, remove the desired volume of conditioned cell culture medium with a pipette and transfer the medium to clean polypropylene microcentrifuge tubes. Centrifuge the medium at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge to remove any cells or cellular debris. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes. These samples are ready for the assay. Alternatively, clarified medium samples can be aliquoted and stored at -80 degrees C for future analysis. Avoid multiple freeze-thaw cycles. Frozen samples should be allowed to thaw on ice just prior to running the assay. Thawed samples should be clarified by centrifuging at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge prior to analysis to prevent clogging of the Luminex™ probe and/or filter plate. Follow the assay procedure provided with the kit for appropriate dilutions.

Answer Id: E12642

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My sample lysates were made in RIPA buffer. Can I test these samples with your ELISA kits?

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Yes, you can. The composition of the traditional 1X RIPA buffer is very similar to that of our Cell Extraction Buffer (Cat. No. FNN0011). Cat. No. FNN0011 is frequently used to prepare lysates for testing with our ELISA and Luminex™ kits. Our NP-40-based Cell Extraction Buffer (Cat. No. FNN0021) is also used. We recommend diluting lysates made with Cat. No. FNN0011 at least 10-fold in order to lower the SDS concentration to less than or equal to 0.01% (v/v) before adding the samples to the ELISA or Luminex™ assay.

Answer Id: E12625

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How does the Luminex™ xMAP™ technology work for measuring more than one analyte in the same well?

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Luminex™ xMAP™ technology is based on polystyrene or paramagnetic microspheres, or beads, that are internally dyed with red and infrared fluorophores of differing intensities. Each dyed bead is given a unique number, known as a “bead region”, allowing the differentiation of beads. For Novex™ multiplex immunoassay kits, individual bead sets are then coated with a capture antibody qualified for one specific analyte. Multiple analyte-specific beads can then be combined in a single well of a 96-well assay to detect and quantify multiple targets simultaneously, using one of the Luminex™ instruments for analysis. We offer multiplex assays using either polystyrene or paramagnetic beads. Please watch this video to learn how to measure multiple proteins simultaneously using the Novex™ multiplex bead-based kits on the Luminex™ instrument platform - https://www.youtube.com/watch?v=kEdLGcGXrs4&feature=youtu.be.

Answer Id: E12636

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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?

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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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How do I process cerebrospinal fluid (CSF) for use on the Luminex™ assay platform?

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All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. However, CSF has relatively low viscosity, and unless there is presence of an infected state (abundance of WBCs), it should not require clarification and can be directly applied to the assay. Dilute 2-fold with Assay Diluent provided in the Neuroscience Buffer Kit (Cat. No. LNB0001).

Answer Id: E12646

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During data analysis, the beads fall below or to the lower left of their bead region on the bead map. Why is this?

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Here are possible causes and solutions for this issue:

This usually indicates that the beads have been photobleached. This problem can also be caused by exposing the beads to organic solvents. Unfortunately, the assay will have to be repeated because the beads cannot be restored. The beads must be protected from light and organic solvents.
Alternatively, the instrument may be off in its measurements or you may have a calibration issue. Call the manufacturer for a service appointment.

Answer Id: E12662

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What is the MAGPIX™ System?

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The MAGPIX™ System is a compact benchtop instrument with a multiplex capability of up to 50 analytes. It is a robust and cost-effective multiplexing tool. Streamlined startup and shutdown protocols, and minimal maintenance requirements, make the system easy to use-ideal for both new and experienced users. It features simple out-of-the-box setup and interactive software. The MAGPIX System analyzes magnetic beads immobilized with a magnet, excites the beads using light-emitting diodes (LEDs), and then detects and analyzes the beads using a CCD camera.

Answer Id: E12639

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I got a poor standard curve after my ELISA. Why is this?

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Here are possible causes and solutions:

Improper preparation of standard stock solution.Dilute the lyophilized standard as directed on the vial label, only with the standard diluent buffer or a diluent that most closely matches the matrix of your sample.
Reagents (lyophilized standard, standard diluent buffer, etc.) from different kits, with either different analytes or different lot numbers, were substituted. Never substitute any components from another kit.
Errors in pipetting the standard or in subsequent steps. Always dispense into wells quickly and in the same order. Do not touch the pipette tips on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device.

Answer Id: E12633

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