I need a protocol to shear flagella off E.coli O157:H7. I have been using a protocol online that is basically resuspending an overnight culture in a syringe and then, spinning it down to acquire the supernatant. This supernatant should ideally contain the flagella, is then precipitated with TCA.
When I've run the sample with SDS-PAGE, the band corresponds to the molecular weight of the flagella. However it is very dilute and I could not get my anti-fliC O157:H7 to detect the flagella in the western.
Any suggestions?
Hi Liyana,
You have two problems as far as I can tell:
1. Low yield of flagella
2. No binding of flagellar ab in western
These are two separate issues.
Firstly, to increase the yield of flagella:
- grow the bacteria in a larger culture volume to a higher density.
- sometimes flagellar expression is repressed in rich media so you may need to use supplemented minimal media
- it is really important to shear the flagella thoroughly by 100s of passes between syringes through a narrow cannula otherwise all your flagella centrifuge out with the cell debris.
Take a look at the following:
J. Bacteriol Vol 182 p 5218
J Bacteriol Vol 161 p 836 and references therein.
Secondly, the western blot problem. This could be due to a multitude of issues.
Is your transfer ok? Try it in a dot blot to ensure that the antibody is capable of binding. Make sure you have the right secondary ab.
Is the anti-flagellar ab capable of working in Wblot or is it optimised for ELISA or other technique?
There should be lots of other Abs you can try.
Are you certain that you have the right E. coli strain-anti flagellin combo?
Hope this helps!
Deepan
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