I think some of you might be familiar with studying several pathways and you check proteins expression by western blot,
did you face issues with selecting endotoxin-free reagents problem? In my lab, we want to seed cells on top of ECM. But no vendors are using the reagents we want to use (for coating) in endotoxin-free situation how did you remove them on your own? if not removed how do you overcome (like do you measure the endotoxin by LAL endotoxin assay and specify the EU/ml level and mention it on your data and move forward with your pathway study for example)?
Thank you
Polymyxin B spin columns are one way of removing any contaminating endoxtoxin in a lot of preparations. However, you would need to determine whether your reagents would be compatible with such and approach.
Dear Daassi
which are the specific reagents that you would like to clean from endotoxin?
Other powerfull coloums for remove endotoxin from solution are endotrap (http://www.hyglos.de/en/index.html) but of course they work for solution and relativelly small volume.
Thanks Adam, we are trying the PMB indeed, however it's recommended for studying other purposes because as you know it binds with endotoxin but does not remove them.
however, in my experiment I look at signaling pathways I cannot add this antibiotic binding (BMP) because it will also interfere with the signaling pathway I want to look at (endotoxins have inflammatory effect and can easily mislead my results)...In fact, I want to remove the endotoxin totally from the reagent not just bind them and keep them in place
Dear Martinelli,
thank you for the recommendation of this interesting kit. In fact, we tried a resin column from different company. it has a broad range that goes to 0.005–1 EU/ml am not sure about endotrap though, I do appreciate it if you could let me know much is the range?
http://www.thermofisher.com/order/catalog/product/88270?gclid=Cj0KEQjw9b6-BRCq7YP34tvW_uUBEiQAkK3svRJRDKl-4to0QO5pMnS8sf6B8gpn7gJDo4ztKKt8XjcaAsNN8P8HAQ&s_kwcid=AL!3652!3!85589465054!b!!g!!_inurl:_feed_label:protein%20analysis&ef_id=VdipZgAAAA6gH8sW:20160907175524:s
currently, we face issues with the fact that our reagent (protein) has actually a high level of endotoxin contamination apparently and that the values we found in EU/ml overshot the standard curve when measuring the by LAL assay, did you try to measure endotoxin after endotrap? am not sure if you did this experiment? but you need to make dilutions of the reagent before you run the LAL and we kept trying several dilutions ...
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