Could anyone give me suggestion about the kit for quantification of endotoxin level in protein and also the kit for endotoxin removal with reasonable price and good sensitivity (
The Thermo Scientific Pierce LAL Chromogenic Endotoxin Quantitation Kit measures the amount of endotoxin in a protein, peptide or antibody sample using the Limulus Amebocyte Lysate (LAL) assay.The endotoxin concentration in a sample is measured using the Pierce LAL Chromogenic Endotoxin Quantitation Kit via a chromogenic signal generated in the presence of endotoxins. Samples can be measured on a microplate absorbance reader at 405nm. A standard curve is created using the E. coli endotoxin standard included with each kit to calculate endotoxin levels as low as 0.1 EU/mL, where one endotoxin unit/mL (EU/mL) equals approximately 0.1ng endotoxin/mL of solution. Protein and antibody samples can be assayed in about 30 minutes. Determining endotoxin levels is important to assess the efficiency of endotoxin removal methods and prevent endotoxic shock, inflammation and/or sepsis in tissue culture cells and animals injected with endotoxin contaminated proteins
We are measuring the endotoxin activity by using the chromogenic kinetic Limulus-Amebocyte Lysate (LAL) assay from Charles River (http://www.criver.com/products-services/rapid-micro/lal-reagents-accessories/kinetic-chromogenic-lal). The assay has a sensitivity of 0.005 - 50 EU. It is not the cheapest one, but it works for a different range of samples, but you have to take care of the sample preparation.
There are also kits available using a recombinant factor c (http://www.lonza.com/campaigns/pharmabiotech/pryogene-rfc-evaluation-program.aspx) or a phage technology (http://www.hyglos.de/en/products-services/products/endotoxin-detection/endolisar.html). They should be less sensitive for interferences.
We just started to measure some samples with this "new" kits.
I used Clear Coli to produce a fusion protein with less LPS, and I measured endotoxin level with LAL Pierce, but after a 100 x dilution my samples are still out of range (0- 1 EU/ml), so I needed more dilution and a best protein production and a more sensitive test because LAL Pierce is only linear from 0-1 EU/ml , anyone have a good suggestion to my problems, thanks.
I am trying Clear Coli cells too. The lucigen suggested the glassware that has been used previously for bacterial cultures may be contaminated with endotoxin even if there are no viable bacterial cells. Baking at >250°C for 1 hour is effective at destroying this residual endotoxin. The medium you use for cell culture should be endotoxin-free water (we have filters on our MilliQ water systems). The column and pump you use for protein purification. The LAL test might give you false positive result. You can check the NF-kappaB activation level as the protocol suggested.
I am trying to remove any possible endotoxin contamination using Pierce Hige-capacity endotoxin removal resin (Thermo scientific 88270) at the last step. Will let you know the result.
Did you use a specific kit of Invivogen for NF-kappa B activation (blue 293 cells with SEAP... a bit expensive) or just your own cells and looking for NF-Kappa B activation after adding your sample of suposed LPS free medium?
Also, with the NF-kappaB activation test it is possible to quantitate the EU/ml of endotoxin o r just look for an in vivo effect?
I have tried the columns from PIerce to eliminate LPS and I have eliminate the LPS and my protein, not very useful in my experience.
with the NF-kappaB activation test it is just looking for an in vivo effect.
I use the 10 ml resin and pack my own column. Yes there is always protein loss, I tried to elute protein with PBS (contain 0.4 M NaCl) which was a little bit helpful to decrease some nonspecific binding of your protein.
Thanks everybody for their comments, I will try many things and send my results.
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