The ELISA assay is a rapid method used to detect the amount of a protein of interest in experimental samples. While this process has become increasing automated and less labour intensive, it is still important to carry out these assays with care to maintain accuracy and maximise the usability of your results. The following are a few tips to keep in mind:
Read the instructions. Sit down the day before you plan to carry out the ELISA and read through the instruction booklet and/or protocol. Make sure you have all your reagents to hand.
Familiarize yourself with the equipment. If it’s your first time using the lab plate washer and spectrophotometer, ask a colleague to show you how they are used.
Plan your plate. Many ELISA kits come with a loading template. Write out exactly what samples and what dilutions you will be analysing. You can tick off each sample as you have loaded them.
Don’t waste precious samples. Carry out a trial run of a standard curve and a few of your samples at varying dilutions first. This will give you an idea of the absorbance values for your experimental samples of interest.
Monitor data quality with replicates. Carry out your standard curve and samples in duplicate or triplicate. You should be aiming for an r2 value as close to 1 as possible.
Minimize variability. Take care with your pipetting and make sure your pipettes are properly calibrated.
Coat your plates all at once. To minimize inter-plate variability it can be possible to coat all the plates your kit provides reagents for and store at -20°C until use. Double check the kit insert to make sure this is possible.
Set up an analysis template. Save yourself time and have an excel template prepared to automatically calculate your standard curve and sample protein levels.
Back up your data! Keep at least one electronic copy of both your raw and analysed data. Label each file clearly with the date and analyte. You never know when you may need to revisit the data.
Take your time. Don’t be overly ambitious and perform more than one or two ELISA plates at a time. This will only lead to mistakes, potentially skewing your results and wasting precious samples and expensive reagents.
How much time do you spend thinking about the accuracy of your pipette? Probably not much. It’s one of those things that get brushed aside in the heat of experimentation. Pipetting accuracy though, is critical to successful experiments–especially in sensitive experiments. For example, qPCR relies upon accurate pipetting—calculations depend on having the same amount of […]
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