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I recently purchased a Complex IV activity dipstick assay from your company and had a few questions about data analysis.
The samples used were of whole blood and they were prepared according to the protocol abcam suggested: Making a 1:3 dilution with the extraction buffer, allowing it to sit on ice for 20 min and then spinning it in a microcentrifuge at 13,000-16,000rpm for 20min. The supernatant was removed and 0.3-9ul of the supernatant was added per dipstick
The assay protocol for the Complex IV activity assay was followed as indicated by the kit insert and a dipstick reader was used to analyze each stick.
Since the reader is new, the stick was read by placing the nitrocellulose membrane inside the machine with the wicking pad facing the door.
However, The data I have obtained is confusing to interpret. I am following each mABS of each peak, which I am expecting only two, one for the positive control and another for the activity complex IV. I assume that the complex IV dipstick has a positive control.
I have attached some data I have obtained on whole blood samples using a range of concentrations. The results are puzzling. I am not sure which peak belongs to Complex IV and which peak belongs to the positive control. Is there an approximate range I should expect Complex IV to show up? I understand that perhaps wicking of a sample can be uneven and therefore give variable results but there must be some range I should be expecting the peak.
Asked on Feb 29 2012
Thank you for contacting Abcam
I sent you query to our lab to ask their opinion on your data and I have attached the file edited with whatthey consider are the positive peaks (without looking at the actual dipsticks). The positive peaks are highlighted in green. The signal is very weak, but the ones highlighted in green are the only ones that show increase in activity with higher loading. The dipstick reader will allocate number of peaks. The peak of interest will not always appear in the same number. Whatyou should look for is the peak at a particular position of X. In this case, they need to look at the peak between 460 and 490 (which in some dipsticks is peak 1, in others is peak 2 and in others is peak 3).
When loading blood, we typically don’t load it by the protein concentration, since most of it is going to be hemoglobin present in red blood cells and albumin present in the serum which are not representative of the mitochondria content in blood (present in PBMCs and platelets).You should load by uL of extract (and not by ug). In the graph below, we have the data you should expect by loading 90uL of blood extract, 45uL……in a 1:2 dilution series. The data was obtained with pooled blood from 3 healthy volunteers. Up to 100uL of volume could be loaded on the dipstick. The 90uL loading was achieved by combining 90uL of extract with 10uL of 10x blocking.
For optimal results, it is also important for you to use fresh blood (not frozen) or if this is not possible, blood should at the very least be stored at -80C viable by adding 10% DMSO just like you would on cultured cells). We have only tested the dipsticks with fresh blood and so cannot guarantee their performance on frozen viable blood, but theoretically it should work well. Also for higher quality data it is recommended to isolate PBMCs from the blood and use that in the assay as opposed to whole blood samples.
Answered on Feb 29 2012