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I have some questions regarding the protocol for ab126556:
1. Is 20ul Brdu, as described in the protocol, added to each well regardless of whether the well contains 100 or 200ul?
2. How are the results to be calculated?
3. Please explain the data showed on the example data included in the protocol.
4. Why is it suggested to either measure OD450-OD550, OD450-OD595 or OD450 alone when measuring the absorbance in each well?
Asked on Jun 05 2014
Q1: Is 20ul Brdu, as described in the protocol, added to each well regardless of whether the well contains 100 or 200ul?
A1: Yes. The amount of Brdu is in excess, so it is fine to add the same 20ul of Brdu whether or not you have added a test reagent. However if you do add a test reagent please ensure that every well has 200ul medium as a final volume, even if not every well contains a test reagent. This is becuase more medium in a well will provide more nutrients for the cells, causing them to perhaps proliferate faster or to a greater degree, throwing off your data.
Q2: How are the results to be calculated?
A2: For each experiment, you will have a well treated with Brdu or not treated with Brdu. Just subtract the wells without Brdu from those which were treated with Brdu. -Brdu wells serve as internal control for background. You can run a blank control as mentioned in the protocol as well. However this is just to ensure that there is nothing inherently wrong with the assay. This blank should always have a very low OD such that subtracting it from all wells will have no effect on the data. However if it is a high value then it is a good indicator there is a problem with the assay.
Q3: Please explain the data showed on the example data included in the protocol.
A3: The green bars should be measured with the Y axis on the right. It measures the ratio of wells treated with and without Brdu (+Brdu/-Brdu). The curves with black and white circles should be qantified using the Y axis on the left. The black circles are OD values from wells treated with Brdu and white circles are from corresponding control wells not treated with Brdu.
Q4: Why is it suggested to either measure OD450-OD550, OD450-OD595 or OD450 alone when measuring the absorbance in each well?
A4: OD550 or OD595 readings act as the reference wavelength. A reference wavelength is used to take into account any variations in the data due to the spec itself. However this reference wavelength value is typically very small so not always used.
Jeremy KasanovAbcam Scientific Support
回复于 Jun 05 2014