Cytokine 25-Plex Human Panel - FAQs

The Cytokine 25-Plex Human Panel (Cat. No. LHC0009M) is suitable for use with the Luminex Instrument platform, including the MAGPIX, LX100/200, FLEXMAP3D, and INTELLIFLEX systems. Details about the Luminex instrument platform and associated Protein and RNA assays can be found on our website by searching " Luminex Instruments".

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Here are possible causes and solutions for this issue:

- Check the protocol settings (make sure you select the correct DD settings).
- Check the level of sheath fluid and empty the waste.
- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.

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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.

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This indicates that an incorrect buffer was used for the final step. The Wash Solution provided in the kit must be used for washing the beads and the Reading Buffer should be used for resuspending the beads before loading them into the Luminex instrument. The osmolarity of the solution will impact the size of the bead, and any change in the bead size will alter detection by the instrument.

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Here are some suggestions:

- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.
- Review the instrument settings and make sure they are appropriate for the assay being run (adjustment of needle height, make sure you select the correct bead gates and the correct DD settings).
- Shake the plate before acquisition on the instrument to resuspend the beads.
- Vortex the beads for 30 sec before adding them into the plate.
- Washing: Do not forget to keep the plate for about 2 mins on the Hand-Held Magnetic Plate Washer before emptying the plate.

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This pattern is indicative of a sample matrix effect. Here are some suggestions:

- Confirm that the sample has been clarified and is free of debris and free of lipids (5-10 min centrifugation recommended).
- Confirm that there is at least a 1:1 ratio of sample to assay diluent for serum, plasma samples. For cell lysates or tissue homogenates, confirm that the sample has been diluted appropriately in assay buffer to reduce the concentration of detergent in the lysis buffer to ⋜0.01%. For other sample types, further sample optimization may be required.

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Here are some suggestions:

- It is possible that the levels of your protein of interest fall below the detection limits of the assay. High Sensitivity Multiplex kits are available for most cytokines.
- Qualify your standard curve (look for plateaus, abnormal curve fits, outliers).

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Here are some suggestions:

- Sample optimization may be needed: Dilute the sample with an appropriate diluent and re-run.
- Qualify your standard curve (look for plateaus, abnormal curve fits, outliers).

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To process cell lysates (extract cellular proteins), follow the instructions provided here (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017835_PrepareCellLysates_Procartaplex_PI.pdf).

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We have not specifically tested saliva samples in-house and therefore these instructions are only our recommendation. Saliva contains several proteolytic enzymes. It would be important to centrifuge samples and be sure not to pipet any cellular material or debris into the assay plate. We would suggest adding some anti-protease in the sample (for example, trasylol or aprotinine, 10 to 50 U/mL) to protect the protein from enzyme degradation. You may then centrifuge the samples at 1000 x g at 4 degrees C for 10 mins to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.

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We have not specifically tested oral mucosal transudate samples in-house and therefore these instructions are only our recommendation. Isolate the site around the tooth and insert a piece of periodontal filter paper into the gum pocket around the tooth for 30 seconds. Remove the filter paper and extract 4 times with 50 µL PBS for 5 min each at room temperature. The individual extractions can be combined and analyzed. Dilute 2-fold with Assay Diluent before applying to the assay.

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We have not specifically tested cervical secretion samples in-house and therefore these instructions are only our recommendation. Cervical sponges should be placed on ice immediately upon collection. Samples should be stored at -20 degrees C for up to one week and then stored at -80 degrees C until ready for assay. After thawing, sponges should be weighed and placed into Eppendorf tubes, using forceps cleaned with ethanol after each transfer. Add 200 µL of ice-cold extraction buffer (recipe below) to each tube and incubate overnight at 4 degrees C. The sponges and extraction buffer can then be transferred to microcentrifuge tubes with 0.2 µm cellulose acetate filters and centrifuged at 13,000 rpm for 10 min at 4 degrees C. The eluate can then be tested for cytokine expression.

Extraction Buffer
50 mM HEPES, pH 7.5
1 mM Na3VO4
150 mM NaCl
1 mM NaF
1 mM EDTA
0.1% Tween 20
25 mM EGTA
10% glycerol

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We have not specifically tested bronchoalveolar lavage (BAL) samples in-house and therefore these instructions are only our recommendation. The bronchoalveolar lavage (BAL) should be collected in a sterile syringe and kept on ice until you are ready to analyze it. Alternatively, BAL can be aliquoted and frozen in usable sample sizes (such that exposure to freeze-thaw is limited to one time). 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 2-fold with Assay Diluent before applying to the plate.

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Centrifuge samples at 1,400 rpm for 10 mins at 4 degrees C to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.

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We have not specifically tested synovial fluid samples in-house and therefore these instructions are only our recommendation. 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.

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Collect spleen, lung, brain, kidney, liver, or heart tissue and treat with or without LPS (100 µg, i.p., for 15 mins, 30 mins, 1 hr, 2 hrs, or 3 hrs). Weigh tissue in a 2 mL microcentrifuge tube. Add 500 µL of Cell Lysis Buffer (Cat. No. EPX-99999-000) per 100 mg of tissue. Add one 5-mm Stainless Steel Bead, then assemble tubes into TissueLyser according to the manufacturer’s recommendations. We recommend using 5-mm Stainless Steel Beads from Qiagen (Cat. No. 69989). Homogenize tissue at 25 Hz for 0.5-3 mins as indicated in the table below. Centrifuge the sample at 16,000 × g for 10 mins at 4 degrees C. Transfer the supernatant to a new microcentrifuge tube. Measure the total protein concentration. Dilute samples to 10 mg protein/mL with 1X PBS. To proceed with ProcartaPlex protocol, add 25 µL of Universal Assay Buffer (Cat. No. EPX-11111-000) to 25 µL of the diluted sample to each sample well.

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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.

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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.

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Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to clot for 20-30 mins at 20-25°C. Centrifuge at 1,000 x g for 10 mins at 20-25°C and collect the serum fraction. Alternatively, a serum separator tube can be used following the manufacturer's instructions. Use immediately or store aliquots at -80°C. Avoid multiple freeze-thaw cycles.

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ProcartaPlex multiplex assays, which are based on Luminex xMAP technology, provide a versatile platform that gives users more flexibility and a greater array of options for analyte detection. Whether you are testing for single or multiple analytes, ProcartaPlex multiplex assays deliver accurate analytical performance using efficient, easy-to-follow protocols. Each of these assays has undergone the same development, validation, manufacturing, and quality control standardization we conduct for our ELISAs. Each lot of ProcartaPlex multiplex assays as well as ELISA assays is fully qualified with the appropriate sample type (i.e., species-specific serum, plasma, and cell culture supernatants), and each lot is evaluated based on the following performance characteristics:

Specificity-each analyte is screened to make sure there is no significant cross-reactivity with other analytes in the multiplex test
Sensitivity-each analyte is evaluated for both functional sensitivity (differentiation from background) and lower limit of detection (LLOD)
Precision/accuracy-multiplex assays have good intra-assay precision (<10% CV), inter-assay precision (<10% CV), and lot-to-lot consistency (<20% CV); these values are comparable to or better than most ELISA tests
ProcartaPlex multiplex assays are regularly tested against the matching ELISAs. Therefore, you can switch easily from ProcartaPlex assays to ELISA and vice versa with reliable results. Most of our ProcartaPlex assays use the same antibody pairs as our traditional plate-based ELISAs, resulting in high correlation (R2 > 0.9) between the two assays.

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ELISA is a simple and powerful way to quantify individual proteins specifically in complex samples. The selectivity of ELISA is achieved through the use of qualified single- or double-antibody sandwich technology, and accurate quantitation is achieved through the use of calibrated standards. ELISAs can detect low-level proteins and can be performed in a 96-well format with only 60 minutes of hands-on time. In addition, the results obtained with ELISAs are generally very reproducible. While ELISA has been established as a standard method of protein analysis, multiplexing methods that enable the measurement of multiple analytes simultaneously in a single sample address a number of specific limitations:

ELISA allows for the measurement of only one analyte at a time in a given sample, limiting investigators' increasing need to measure multiple targets in their research studies.
The low available volume of many samples being studied may limit the number of times analyses can be conducted. This is especially true in small animal research, in pediatric testing, and in microplate assays providing limited sample volumes. The ability to assay multiple analytes in a single small-volume sample enables more effective use of each sample.
Difficulties in data interpretation can arise when comparing analyte levels measured by multiple ELISAs, each assay having been performed with different sample aliquots and each susceptible to systematic errors leading to decreased precision and accuracy.
Many analytes require assays with broad dynamic ranges to avoid repeat testing or out-of-range values. Multiplex assays can be designed to have large dynamic ranges for all of the analytes, or ranges tailored to various expected analyte concentrations.

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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 ProcartaPlex 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.

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The Luminex assay is a bead-based immunoassay that uses beads of defined spectral properties conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration 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 incubation on a shaker, the beads are washed by putting the 96-well plate on a flat magnet for 30 seconds, after which the fluid is discarded by flicking the wells or by using an automated plate washer. The magnet is removed, and the beads are resuspended in the detection antibody. Another incubation and wash are followed by the addition of streptavidin–R-phycoerythrin (SAPE). The beads are then washed and are ready to analyze. The Luminex technology is compatible with the following Luminex analyzers:

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

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Here are possible causes and solutions:

Errors in pipetting the standards or samples or in subsequent steps. Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device. Check for any leaks in the pipette tip.
Repetitive use of tips for several samples or different reagents. Use fresh tips for each sample or reagent transfer.
Wells have been scratched with the pipette tip or washing tips. Use caution when dispensing into and aspirating out of microwells.
Liquid transferred from well to well during incubations. Adjust the orbital shaker or check for correct rotator rpm. Peel the adhesive plate cover off carefully.
Incorrect volumes of materials dispensed into the microwells. Follow the protocol for dispensing volumes of reagents. Check calibration of the pipettes.
Standard diluted with the serum, culture medium, or other buffer. Dilute the standard with the standard diluent buffer provided in the kit.
Particulates or precipitates present in the samples. Remove any particulates/precipitates by centrifugation prior to dispensing into the assay.
Dirty microwells: visible debris within or on bottom of microwells. Inspect the microwells and invert the plate to remove debris. Wipe the bottom of the plate with an absorbent tissue after each wash step. Never insert tissue into the microwells.
“Edge effect” due to uneven temperature between the outer-edge wells and the wells in the center of the plate. Seal the plate completely with a cover during incubations, and place the plate in the center of the incubator when 37 degrees C incubation is indicated.

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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.

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Here are possible causes and solutions:

Reagents not at room temperature (approximately 25 plus or minus 2 degrees C) at start of assay. Allow all reagents to warm to room temperature prior to commencing the assay.
Incorrect storage of components, e.g., not stored at 2-8 degrees C. Store all components exactly as directed in the protocol and on labels.
Anti-rabbit IgG HRP or streptavidin-HRP working solution made more than 15 minutes before use in assay. Use the diluted anti-rabbit IgG HRP or streptavidin-HRP within 15 minutes of dilution.
Expired reagents.Check expiration dates upon receipt of kit and use the kit prior to expiration.
Plate read at incorrect wavelength. The correct wavelength to read ELISAs using the TMB substrate is 450 nm.
TMB solution lost activity. Ensure that the TMB solution is clear before it is dispensed into the plate wells. A blue color and/or the presence of particulate matter indicate that the product is contaminated. Please contact Technical Support if this problem is noted. To avoid contamination, we recommend that the quantity required for an assay be dispensed into a previously unused disposable trough for pipetting. Discard any TMB solution left in the trough and do not put it back in the bottle. Avoid contact between the TMB solution and items containing metal ions. Do not cover your plates with aluminum foil or aluminum-coated Mylar sheets because this can cause color development in the absence of HRP.
Attempt to measure analyte in a matrix for which the ELISA assay is not optimized. Contact Technical Support when using alternative sample types.
Wells have been scratched with pipette tip or washing tips. Use caution when dispensing into and aspirating out of microwells.
Incorrect chromogen or stop solution used. Use only the chromogen and stop solution supplied with the kit.
Standard diluent buffer added to all wells rather than the designated wells. Follow the protocol and only add the standard diluent to the designated wells and to the samples where it is required, or to samples producing signals greater than that of the highest standard.
Use of buffer containing azide, which is not compatible with HRP. Avoid the use of azide in the assay.

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Here are possible causes and solutions:

Incorrect dilution of the anti-rabbit IgG HRP or streptavidin-HRP working solution.
Warm the solution of anti-rabbit IgG HRP or streptavidin-HRP (100X) to room temperature, draw it up slowly, and wipe the tip with a laboratory tissue (e.g., Kimwipe tissue) to remove the excess. Dilute only in the HRP diluent provided.
Incubation times extended. Follow incubation times outlined in the protocol.
Incubations performed at 37 degrees C. Perform incubations at room temperature (approximately 25 plus or minus 2 degrees C) when instructed in the protocol.

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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.

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Our customers use a wide variety of plate readers. Some of these can't read absorbances higher than 2 AUFS (Absorbance Units Full Scale), while others can't go beyond 3 AUFS, for example. If you read your ELISA plates after 30 minutes of incubation at room temperature and 1 or 2 A450 values are off-scale, you can shorten the incubation time. For example, some customers find that 20 minutes is the ideal incubation time because the ambient temperature in their lab is higher than approximately 2 degrees F (22 degrees C). In this case, higher temperatures increase the rate of the HRP-driven ELISA. Conversely, if the A450 values you get are not high enough, you can increase the incubation time accordingly.

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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.

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Our Antibody Pair kits include matched, pre-titered and fully optimized coating and detection antibodies, 3 vials of recombinant protein standard, the streptavidin-HRP conjugate concentrate, and a lot-specific technical data sheet. The buffers are not included. The Buffer Kit needed to run the Antibody Pair ELISA must be purchased separately (Cat. No. CNB0011). Note that the 5X Assay Buffer supplied in Cat. No. CNB0011 is used to block your ELISA plates and as a diluent for the standards and samples.

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No. The exact same streptavidin-HRP conjugate supplied in these kits is not available as a stand-alone product. However, it is derived from our ELISA-grade streptavidin-HRP (Cat. No. SNN2004), which we do sell. Remember that the streptavidin-HRP provided in each lot of ELISA or Antibody Pair kits is also lot-specific. So, if you use Cat. No. SNN2004 or another source of streptavidin-HRP, you will have to determine which dilution of SNN2004 works best for you.

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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.

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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.

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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.

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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.

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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.

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Each Antibody Pair kit contains capture (coating) antibody, biotinylated detection antibody, recombinant standard, and streptavidin-HRP. Other reagents required are listed in the Antibody Pair manual included with the kit, and can also be purchased separately (Antibody Pair Buffer Kit, Cat. No. CNB0011; 5X Assay Buffer, Cat. No. DS98200; etc.). The manual also provides a specific procedure and illustrates an example of a standard curve that can be obtained when the specific procedure is followed.

A general procedure is summarized here:
1) Coat the microplate with diluted capture (coating) antibody overnight at 2-8 degrees C; wash the plate.
2) Incubate the standards or samples in the coated microplate; wash the plate.
3) Incubate diluted biotinlyated detection antibody in the plate; wash the plate.
4) Incubate streptavidin-HRP in the plate for 15-45 min; wash the plate.
5) Incubate the plate with TMB substrate for 10-60 min, and then stop the reaction with Stop solution.
6) Read the microplate at 450 nm.
We recommend determining optimal buffer formulations, concentrations, and incubation times for individual applications.

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