How to Use an ELISA Microplate on a Microplate Shaker

Описание: Using a microplate shaker (oscillator) is a standard and critical step in many ELISA protocols to ensure proper mixing of reagents. Correct use enhances assay performance by promoting uniform binding reactions and reducing variation between wells.

I. Purpose of Shaking in ELISA
Shaking serves two primary functions:

Mixing: Ensures thorough and homogeneous mixing of added reagents (samples, antibodies, substrates) within each well, preventing concentration gradients.

Kinetic Enhancement: Increases the rate of interaction between molecules in solution and those immobilized on the plate surface, which can improve binding efficiency, reduce total incubation time, and enhance assay sensitivity.

II. Step-by-Step Operational Guide

A. Preparation

Select the Correct Shaker: Use a dedicated microplate shaker with a flat platform or an adjustable carrier designed to hold microplates securely. Ensure it is placed on a stable, level surface.

Secure the Plate: After adding the required reagent to the plate, apply a plate sealer firmly to prevent evaporation and aerosol formation. For incubation steps longer than a few minutes, sealing is mandatory.

Position the Plate: Place the sealed plate centrally on the shaker platform. If using a shaker with a retaining frame or bracket, secure it to prevent movement.

B. Setting Shaking Parameters

Speed (RPM): A moderate speed is optimal. Typical ELISA protocols recommend a range of 300-600 rpm. Excessive speed (more than 800 rpm) can cause vortexing, increase evaporation risk if the seal is imperfect, and potentially lead to cross-contamination. Start at 400-500 rpm if no specific instruction is given.

Motion: Most protocols use orbital shaking (circular motion). Some shakers offer linear or dual-action motion; orbital is the standard.

Duration: Shaking time varies by step:

During Incubations: Continuous shaking for the entire incubation period (e.g., 60 minutes for antigen-antibody binding) is common and ideal.

After Reagent Addition: If not shaking continuously, a brief pulse of shaking (e.g., 30 seconds to 1 minute at 500 rpm) immediately after adding a key reagent is essential to mix the well contents before the start of a static incubation.

C. Execution

Start the Shaker: Set the desired speed and time. Start the shaker after the plate is securely positioned.

Monitor: During initial use, observe for a few seconds to ensure the plate is stable and not sliding.

Post-Shaking Handling: After the shaking cycle is complete, carefully remove the plate. If proceeding to a wash step, gently remove the sealer to avoid creating aerosols. Before reading absorbance, briefly centrifuge the plate (200-500 x g for 1 minute) to pool all liquid and eliminate bubbles created during shaking.

III. Critical Best Practices and Warnings

Always Seal the Plate: Never shake an unsealed plate. This is a major contamination hazard.

Balance the Load: If shaking multiple plates, balance them symmetrically on the platform to prevent excessive vibration and wear on the shaker motor.

Avoid Condensation: If incubating at a temperature different from the shaker environment (e.g., a 4°C incubation on a room-temperature shaker), ensure the plate is fully sealed to prevent condensation under the sealer, which can dilute wells.

Consistency is Key: Use the same shaking speed, time, and motion type for all plates within a single experiment to ensure comparable results.

Protocol First: Always defer to the parameters specified in your specific ELISA kit protocol. Some kits may recommend static incubation or provide precise shaking instructions.

IV. Troubleshooting

High Well-to-Well Variation: Inconsistent shaking or an unsecured plate can cause this. Ensure the plate is held flat and stable.

Low Signal/High Background: Insufficient shaking can lead to poor binding or uneven substrate reaction. Ensure shaking is continuous or of adequate duration.

Evaporation in Edge Wells: This indicates an imperfect seal. Use high-quality adhesive plate seals and apply pressure evenly around the rim.

Conclusion
Proper use of a microplate shaker is a simple yet vital technique for optimizing ELISA performance. By securing and sealing the plate, applying moderate orbital shaking (300-600 rpm), and maintaining consistency across experiments, you significantly enhance reagent mixing and molecular interactions, leading to more robust, reproducible, and sensitive assay results.