Apologies for the slightly frivolous language
– technically peptidoglycan is *an*
endotoxin, as there’s a separate entity also commonly referred to as
endotoxin which is the LPS/LOS component of the cell wall. Peptidoglycan is
also an endotoxin (or at least quite a few researchers consider it to be one)
which is why I would like to correct my previous answer.
You could try BacTx from Immunetics, for
pure peptidoglycan assay. Not sure if it’s fully commercial or not but
they might send you a sample.
Since endotoxin contamination and
peptidoglycan contamination usually go hand in hand, the endotoxin assay should
be indicative of PG presence as well. The crab blood assay (I am not kidding!)
is probably too sensitive as it is designed to detect minuscule amounts of
contamination.
Artem
---
When the Weasel comes to give New
Year's greetings to the Chickens no good intentions are in his mind.
From:
Sent: Thursday, February 26, 2009
6:57 PM
To: '
Cc: [log in to unmask]
Subject: RE: [ccp4bb] Questions
regarding Peptidoglycan-binding protein
Look up a standard endotoxin assay
(usually an immunoassay) because that’s exactly what endotoxin is J
Artem
---
When the Weasel comes to give New Year's
greetings to the Chickens no good intentions are in his mind.
From: CCP4 bulletin
board [mailto:[log in to unmask]] On
Behalf Of
Sent: Thursday, February 26, 2009
5:53 PM
To: [log in to unmask]
Subject: [ccp4bb] Questions
regarding Peptidoglycan-binding protein
Dear all,
Thank you in advance for your expert opinions.
I am working on a peptidoglycan (PG)-binding protein, which is composed of two
functionally identical domains. Interestingly, when subjected to gel filtration
chromatography (Superdex 200), both full-length protein and the single domains
migrate as a major peak tailed with a broad shoulder. SDS-PAGE indicates the
expected MW for both samples from the major peak and the shoulder, however,
with the presence of smears at high molecular weight. Also, after all fractions
from both the major peak and the shoulder were combined and incubated at 4 C
over 48 hours, the sample was applied to gel filtration again; this time the
shoulder actually disappeared (or shifted), resulting in a nice symmetric peak
(with the same elution volume as the major peak).
My guess is that the protein expressed in E. coli may already specifically or
unspecifically associate with PG fragments from cell wall. My question is: how to detect PG fragments in my protein sample?
I have tried denaturation-wash-renaturation steps, but I want to know if I have
completely got rid of the possible "contaminants".
Thank you for suggestions.
Joe