In order to perform a colorimetric assay (by spectrophotometer), generally two types of protocol were described in literature, i.e. standard assay vs micro plate assay. In common perspective, i do understand that, reagents and samples will be minimized by micro plate assay compared to standard assay. Apart from this, what are the other crucial factors, which are to be considered while doing micro plate assays so as to maintain the accuracy. As beginner, i scared about micro plate assay that, error will be added due to the process of dilution, which is mandatory for micro plate assay. (In my case, i need to quantify the total protein concentration from serum as well as tissue samples by Bradford Assay).
Thanking you in advance for sharing your valuable experience.
Hi Annadurai, to begin with, you can't escape dilution/pipetting errors no matter what colorimetric assays you do. The advantage of doing colorimetric analysis using a microplate is that you can do the assay in duplicate (or triplicate...etc) and take the average of the values.
Also an advantage of the microplate assay is that you can generate a standard curve of protein concentration at the same time you measure your samples.
I agree with Nikonov, The best approximation of the ideal situation is the ideal solution.
It is true that using microplate assays saves a lot of chemical, time and money. It also helps in measuring multiple times the replicates of the same sample. For end point reaction where the sample is incubated and reading is taken, using multichannel pipettes will help you. In case of kinetic reactions (enzyme activity measurement) add all the reagents except the final volume which you can add in the last minute using the multichannel pipette (5-8) depending on your requirement. Also you will need a microplate reader. As already told measuring multiple times will not only reduce the error due to instrument, by replicating a sample, error due to pipetting will also be minimised.
If your pippetting skill is not so good then the variation tends to be much bigger than the standard method. Once you are cnfident with your pippetting skill then microplate method can save you a lot of sample for downsteam tests.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods