Why did you need to wash the wells after every step?

Washing the microplate wells after every step in various biochemical and molecular biology assays such as ELISA (Enzyme-Linked Immunosorbent Assay), DNA/RNA isolation, PCR, and Western Blotting is an essential part of the experimental protocol. The purpose of washing steps is to remove residual reagents, reaction components, or any contaminants that might interfere with subsequent steps or affect the accuracy and specificity of the assay.

Here are a few reasons why washing steps are necessary:

Removal of unbound substances: Washing steps after adding reagents or antibodies help to remove any unbound substances, such as excess antibodies, unspecific binding molecules, or reaction components that are not captured or bound to the solid phase. This ensures that the detection signal is specific and free from background interference.

Minimizing cross-contamination: Washing steps effectively reduce the risk of cross-contamination between samples. This is particularly important when multiple samples are processed simultaneously, as it prevents carryover of reaction components or contaminants from one well to another. Washing helps maintain the integrity and accuracy of individual sample results.

Efficient removal of interfering substances: Washing is necessary to remove substances that might interfere with subsequent steps. For example, in an ELISA assay, washing helps eliminate unbound conjugate-antibody complexes, thereby reducing background color development and enhancing the specificity of the colorimetric reaction. In PCR, washing removes nucleotides, primers, and polymerases from previous amplification cycles, minimizing the risk of carryover contamination and ensuring accurate amplification in the next cycle.

Standardization and reproducibility: By following standardized washing procedures, researchers ensure that the conditions are consistent across experiments and between different laboratories. This consistency is critical for obtaining reliable and reproducible results.

The washing procedure may involve using a multichannel pipette or an automated plate washer to dispense and aspirate the wash buffer. The wash buffer is typically a buffered saline solution that resembles the composition of the original buffer used in the assay, but does not contain any reaction components.

Adequate washing without over-washing is crucial. Over-washing can result in the loss of essential components bound to the solid phase and affect the sensitivity of the assay. Therefore, washing protocols are carefully designed to achieve optimal removal of unwanted substances while preserving the integrity of the immobilized molecules or analytes of interest.