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FNN0011

Product FAQ

What is the MAGPIX™ System?

Answer

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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Product FAQ

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?

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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Product FAQ

How do I process oral mucosal transudate for Luminex™ assays?

Answer

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.

Answer Id: E12649

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Product FAQ

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

Answer

Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to sit at room temperature for 15-30 min to clot. Spin at 1,000-2,000 x g for 10 min in a 4 degrees C refrigerated centrifuge to separate the cells. 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 serum is to be analyzed at a later date, apportion it into 0.5 mL aliquots and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When possible, avoid the use of hemolyzed or lipemic sera. We recommend that upon thawing the samples be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered, prior to analysis, to prevent clogging of the filter plates and/or probe. Follow the assay procedure provided with the kit for appropriate dilutions.

Answer Id: E12640

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Product FAQ

After I added the chromogen reagent to the plate, I incubated the ELISA as suggested in the manual, but the A450 of the highest standard was higher than what my plate reader can read. What should I do?

Answer

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.

Answer Id: E12629

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Product FAQ

I see very weak to no color development after my ELISA. What happened?

Answer

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.

Answer Id: E12632

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Product FAQ

How do I process saliva samples for the Luminex™ assay platform?

Answer

We have not specifically tested saliva samples in-house. 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 treat the sample as you would a supernatant; follow the procedure described for cell culture supernatant.

Answer Id: E12650

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Product FAQ

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

Answer

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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Product FAQ

How do I develop a sandwich ELISA using Antibody Pairs?

Answer

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.

Answer Id: E12616

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Product FAQ

Why should I consider switching from ELISA technology to multiplexing?

Answer

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.

Answer Id: E12637

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Product FAQ

I ran out of the streptavidin-HRP that came in your ELISA (or Antibody Pair) kit. Can I get more?

Answer

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.

Answer Id: E12626

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Product FAQ

What extraction reagents are recommended for efficient Mouse tissue analysis?

Answer

We have 5 different cell and tissue extraction buffers suitable for preparing mouse cell and tissue extracts. These buffers can be used to extract cells and tissues from many other species as well. The exact compositions of all of our buffers are proprietary, but they are similar to those described by many researchers.

Four of these buffers can be used to prepare extracts which can be analyzed with our ELISA and Luminex kits and by Western blotting. Our Cell Extraction Buffer (FNN0011) contains extra phosphatase inhibitors and resembles the RIPA formulation that many people use. Our Tissue Extraction Reagents I (FNN0071) and II (FNN0081) contain different concentrations of NaCl and different surfactants, but are otherwise similar to each other. For those who prefer using an extraction buffer containing the detergent NP-40, we have our NP-40 Lysis Buffer (FNN0021). Finally, we sell a Denaturing Cell Extraction buffer (FNN0091) which contains 3 detergents and a chaotropic agent. Extracts prepared with FNN0091 can be analyzed with our ELISA kits and by Western blotting only. These buffers do not contain protease inhibitors, which the investigator should add right before use.

Answer Id: E5224

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Product FAQ

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

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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Product FAQ

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

Answer

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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Product FAQ

I am observing liquid on the XY platform tray in the instrument after the run is over. What could have caused this?

Answer

This may originate from the probe height being set too low, and the pressure from the fluid return is forcing liquid through the membrane. However, remember that as long as the required 100 beads are being read in a reasonable time frame, the sample is being read correctly. If there is difficulty reaching 100 beads, liquid may have leaked out prior to reading the sample. In this case, stop the run and check the plate for leakage. If leakage is found, remove the Wash Solution on the vacuum manifold and completely dry the bottom of the plate. Add fresh Wash Solution and shake. Continue the run from where it was stopped.

Note: Refer to the appropriate instrument hardware manual for instructions to check and to reset the sample probe (sample needle) height.

Answer Id: E12655

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