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

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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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Can I store Luminex™ assay plates overnight and read them the next day?

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If the plate cannot be read on the day of the assay, cover and store the plate in the dark overnight at 2-8 degrees C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate, bring the plate to room temperature on an orbital plate shaker (still protected from light). Aspirate the Working Wash Solution from the stored plates and add 100 μL of fresh Working Wash Solution. Place the plate on an orbital shaker for 2-3 min at 500-600 rpm prior to analysis.

Answer Id: E12653

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My filter plate is leaking. What should I do?

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Here are some possible causes and solutions for this problem:

Placing the plate on an absorbent material both at the bench and on the shaker (which results in wicking when there is contact between the absorbent material and the opening on the bottom of the well funnel). Rest the plate currently in use on top of a second filter plate so that the filter plate currently in use only comes into contact with a nonabsorbent surface. This will also help locate any potential leaks.
Prior to incubations, use a Kimwipe™ tissue and lightly press up on each well to dry off the bottom of the plate. Putting any vertical pressure on the plate during the incubations (using either tape or clamps).
Use clamps that fit around the sides of the filter plate to secure the plate to the shaker during incubations. Subjecting the plate to vacuum pressure greater than 5 mm Hg, even for a moment, which either tears the filter membrane or creates an opening that exceeds the design limits for volume retention.
Prevent any vacuum surge by opening and adjusting the vacuum on the manifold before placing the plate on the manifold surface and checking the vacuum with a test plate (i.e., not the plate used for the assay). For our assays we recommend the setting not exceed 5 mm Hg. Puncturing the filter membrane with pipet tips, by inserting the tips all the way into the wells during reagent loading.
Pipet solutions along the sides of the wells, rather than deep into the wells. Separating the soft (opaque) plastic bottom from the hard (clear) plastic top of the filter plate before starting, which compromises the integrity of the seal even if the pieces appear to fit back together properly. If you accidentally separate these two layers of the plate, the plate should be discarded and a fresh plate used instead.

Answer Id: E12654

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How do I process urine sample for use on the Luminex™ assay platform?

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Use only freshly collected urine samples. Dilute 2-fold with the Assay Diluent provided in the kit. The final dilution of the sample will be 4-fold, and all results should be multiplied by 4. As needed, clarify samples by centrifugation (14,000 rpm for 10 min) and/or filter them prior to analysis to prevent clogging of the filter plates and/or probe.

Answer Id: E12644

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

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

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

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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.

Answer Id: E12626

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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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My filter plate has clogged wells. Do you have any suggestions?

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If wells are clogged after the first incubation with the standards:

When washing a partial plate, the vacuum may not be complete because air is being drawn through the empty wells. We recommend covering the plate with Parafilm™ film to create a seal, which will in turn raise the pressure high enough to empty the wells. If there is a small clog, it may be difficult to see it even though it is enough to prevent aspiration. For this, we suggest using the tip of a 15 mL conical tube to gently rub against the tip of the well bottom opening, going from left to right. This will dislodge small clogs only. Other ways to unclog a well are to apply pressure from above the well using a gloved finger or thumb with an absorbent paper towel under the plate, or unplugging the drain hole using a large syringe needle.

Answer Id: E12656

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I have an elevated optical density (OD) reading for my ELISA standard. What went wrong?

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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.

Answer Id: E12631

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

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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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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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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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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?

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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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I bought one of your Antibody Pair ELISA kits, but there were no buffers included. How should I set up and run the ELISA?

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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.

Answer Id: E12627

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