Lysyl Oxidase Activity Assay Kit (Fluorometric) (ab112139)

Theoretically, azide should be compatible with ab112139 Lysyl Oxidase Activity Assay Kit (Fluorometric), though I am sorry we have not directly tested this so cannot provide data to confirm this.

Compounds and buffer components that may interfere with the assay include DTT, glutathione (reduced form: GSH), β-mercaptoethanol, some detergents (such as Brij-35, Tween-20 and NP40), NADH and NADPH.

The laborotory uses 500 uM BAPN from TCI, the cat# is A9754, and the cas# is 2079-89-2. In general the laboratory would recommend between 0.1 - 1 mM as most publications use BAPN concentrations in this range. Please see some example references attached.

Please note that this kit is not an ELISA kit but an activity assay kit.

With regards to the standard used, when the kit was first developed, the lab used isolated LOX from pig heart, which is not commercial available. The kit is now tested using recombinant human LOXL2 from R&D Systems-

http://www.rndsystems.com/pdf/2639-AO.pdf

Frozen samples are suitable for use with this kit.

For cell lysates, the cells should be collected and then homogenized in PBS. After spinning down the cells, the supernatant will be used in the assay. 50 uL of supernatant will be added per well of the assay plate, and follow step C.1. in the protocol (page 10).

I can confirm that Solid Black micro plate was used in experiment with top-reading instrument.

The clear bottom black and solid black plates can be selected based on experimental set up and instrument.

1. Should one use fresh or frozen samples? --Both are OK as long as there is LOX in the sample
2. Are tissue extracts OK?--Yes.

1) Regarding the standard serial dilutions, you are correct it should be 0.04 ug to 4 ug. This has been updated in our online protocols, but older copies of the protocol may still incorrectly say 0.04 ng. Please make the standards starting with 0.04 ug.

2) You can use other LOX standard protein, but we suggest using the R&D product as that is what the lab uses. Searching on http://www.biocompare.com may give you other options of LOX standard. When preparing the standards, use PBS with 0.1% BSA or the buffer in your test sample. The assay buffer contains LOX substrate in it, so don't use it for your sample and/or standard dilution.

3) For the microplate, we suggest using Greiner Bio-one cat #655079.

I contacted the scientists in the lab about this question, and we have not tested the kit with samples containing protease inhibitors, but theoretically the kit should be compatible with protease inhibitors. It should be fine to add these to your samples prior to use with the kit.

Thank you for contacting us.

The LOX activity is expressed as Fluorescent RFU units. Please check the following publication for reference;

http://www.molecular-cancer.com/content/9/1/206

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.