At point 10 of the assay protocol (page 10) the samples are centrifuged at 10,000 x g for 3-5 minutes. Then at point 11, 12 and 13 a volume of 10ul of the respective sample or control specific solution is added. The protocol does not mention anything about taking off the supernatant after centrifugation. If the 10ul are added to the preparation including pellet, we are wondering what the centrifugation is used for?
The reason of the 10000 x g centrifuge step # 10 is to effectively “sequester” the debris in a tight pellet at the bottom of the tube keeping it separated for the most part from the supernatant fraction for the rest of the assay. If the customer prefers, the supernatant from step 10 can be transferred to other tubes at this point but this would require another set of vials and is not necessary.
Steps 14 and 17 each call for a brief one second vortex to assure mixing. We have found it possible to mix the TCEP and XOF Reagents without fully suspending the pellet. If some of the pellet is suspended it isn’t an issue as both reagents will still be able to react effectively after a quick 1 second mix. In the event that some of the pellet becomes dislodged the whole reaction mix is again spun hard at 10,000 xg (step 19) just before transferring the mix for reading at the end of the assay. In any case, the main reason we do not include instructions to harvest the supernatant after step 10 is to save time and avoid confusion with having to create another large set of tubes for transfer.