Tips & Tricks: Preparation of Reagents for MILLIPLEX^® Multiplex Assays

Looking for some assistance with the preparation of reagents for MILLIPLEX^® multiplex assays? Check out these tips for general preparation techniques and specific advice for wash buffer, quality controls, standards/calibrators, serum matrix, and beads.

• General Reagent Preparation Techniques
• Wash Buffer
• Quality Controls
• Standards/Calibrators
• Serum Matrix
• Antibody-Immobilized Beads
• Request Information

General Reagent Preparation Techniques

Our MILLIPLEX^® multiplex assay protocol procedures are optimized for the best data results. Consequently, protocols can vary from kit to kit. It is important to read the entire protocol before proceeding.

General reagent preparation techniques for MILLIPLEX^® kit protocols include:

• Deliver precise volumes of solvent when reconstituting lyophilized products. Variations of even a few microliters can significantly affect quantitation.
  
• Do not mix or substitute assay reagents with those from other lots or sources.
• If leftover reagent lots match and the reagents have been kept at the appropriate storage conditions, they can be used in combination until the expiration dates.
• Serum matrix, bead diluents, and wash and assay buffers from other kits can be used/combined if the catalog numbers of these components match in the protocols for the kits being used.

Wash Buffer

• Bring the 10X wash buffer to room temperature and mix to bring all salts into solution.
• Dilute 60 mL of 10X wash buffer with 540 mL deionized water.
• Store unused portion at 2-8 °C for up to one month.
• If more wash buffer is required, see protocol sections “Reagents Supplied“ or “Replacement Reagents“ for the appropriate catalog number.

Quality Controls

Quality Controls (QCs) are included in every MILLIPLEX^® multiplex kit to qualify assay performance.

• Before use, reconstitute QC 1 and QC 2 with 250 µL deionized water.
  
• Invert the vial several times to mix and vortex.
• Allow the vial to sit for 5-10 minutes and then transfer the controls to appropriately labeled polypropylene microfuge tubes.
• Unused portion may be stored at ≤ -20 °C for up to one month.
• Refer to lot-specific QC range sheet for appropriate QC concentration ranges.
  • QC range sheets are included in each MILLIPLEX^® kit box or can be found on MILLIPLEX^® product detail pages.

Standards/Calibrators

Figure 1 shows an example of a standards/calibrators preparation.

• After hydration/reconstitution, all standards and controls must be transferred to polypropylene tubes.
• During the preparation of standard curves, thoroughly mix each higher concentration before making the next dilution.
• Use a new pipette tip with each dilution.
• The standards prepared by serial dilution must be used within one hour of preparation.
  • Discard any unused standards except the standard stock.
  • The standard stock can be stored at ≤-20 °C for one month or at ≤-80 °C for more than one month.
• The quality of the standard curves can be determined by the % recovery of the standards and the QC values.

Serum Matrix

This serum matrix step is required for serum or plasma samples only.

• In most cases, add 1.0 mL deionized water to the bottle containing lyophilized serum matrix.
  • Refer to the specific kit protocol for detailed instructions on Serum Matrix preparation.
• Mix well. Allow at least 10 minutes for complete reconstitution.
• Leftover reconstituted serum matrix can be stored at ≤-20 °C for up to one month.

Kits designed for non-serum/plasma samples (e.g., urine, CSF) or samples that require a significant dilution (at least 1:20) do not require serum matrix. For non-serum/plasma samples, the appropriate medium (e.g., cell culture medium) should be added instead of serum matrix.

• In the absence of an appropriate medium or when using a blank, assay buffer can be used.
• For cell/tissue homogenates, the final cell or tissue homogenate should be prepared in a buffer that has a neutral pH, contains minimal detergents, or strongly denaturing agents, and has an ionic strength close to physiological concentrations.
• Normalize the sample protein concentration with lysis buffer according to the protocol.
  • For example, dilute sample to 0.8 µg/µL with lysis buffer. Then dilute the 0.8 µg/µL sample 1:1 with kit assay buffer. The matrix here is then a 1:1 dilution of lysis buffer with kit assay buffer and the protein concentration is now 0.4 µg/µL.

Why Use Serum Matrix?

Blood is a complex matrix, containing proteins that may interfere with the accurate measurement of your specific analytes. Using an optimized serum matrix in the standard curve when measuring analytes in serum/plasma samples more accurately simulates the conditions of the native analytes, significantly improving the accuracy of measurement.

Antibody-Immobilized Beads

• For individual vials of beads, sonicate each antibody-bead vial in an ultrasonic waterbath for 30 seconds. Vortex for 1 minute.
  • Sonicator probes should not be used.
• Follow the protocol to prepare each antibody bead vial and add antibody beads to the mixing bottle and bring final volume to 3.0 mL with bead diluent.
• Vortex the mixed beads well.
• The antibody-immobilized beads are light-sensitive and must be protected from light at all times.
  • Cover the assay plate containing beads with an opaque plate lid or aluminum foil during all incubation steps.
• Any unused mixed antibody-immobilized beads may be stored in the mixing bottle at 2-8 °C for up to one month.

Don’t want to mix beads? Contact your sales representative about receiving premixed beads for select kits. Premixed beads are background tested and assessed for the appropriate bead regions.

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For Research Use Only. Not For Use In Diagnostic Procedures.