Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Good antibody for E. coli surface marker


  • Please log in to reply
2 replies to this topic

#1 peanut142

peanut142

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 11 February 2010 - 08:11 PM

Hey guys,

I am doing phagocytosis assay with macrophages and my GFP-E. coli. In order to distinguish between intracellular and extracellular bacteria after infection, I need to label the extracellular bacteria with an antibody. The protocol is as below:

1. co-incubate macrophages with E. coli for 2h
2. wash the macrophages 2-3 times with PBS and fix with 3.7% formaldehyde (Cells are not permeablised here as I only want the E. coli antibody to recognise extracellular e. coli)
3. Block with 5% FBS/PBS
4. Incubate cells with Primary antibody against E. coli o/n at 4 degree
5. Wash with PBS twice and incubate the 2nd antibody for 1h at room temperature
6. Wash with PBS twice and permeablise macrophages with Triton
7. DAPI staining and then mount

I got the GFP-e.coli from someone and she told me that it was a DH5alpha. Do you guys know any good antibody that recognise the surface of DH5alpha without permeablising the bacteria? Any suggestions are welcome. Thanks a lot in advance!

#2 fishdoc

fishdoc

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 272 posts
2
Neutral

Posted 12 February 2010 - 05:10 AM

Hey guys,

I am doing phagocytosis assay with macrophages and my GFP-E. coli. In order to distinguish between intracellular and extracellular bacteria after infection, I need to label the extracellular bacteria with an antibody. The protocol is as below:

1. co-incubate macrophages with E. coli for 2h
2. wash the macrophages 2-3 times with PBS and fix with 3.7% formaldehyde (Cells are not permeablised here as I only want the E. coli antibody to recognise extracellular e. coli)
3. Block with 5% FBS/PBS
4. Incubate cells with Primary antibody against E. coli o/n at 4 degree
5. Wash with PBS twice and incubate the 2nd antibody for 1h at room temperature
6. Wash with PBS twice and permeablise macrophages with Triton
7. DAPI staining and then mount

I got the GFP-e.coli from someone and she told me that it was a DH5alpha. Do you guys know any good antibody that recognise the surface of DH5alpha without permeablising the bacteria? Any suggestions are welcome. Thanks a lot in advance!




I don't know of any particular antibodies. E. coli is so commonly used, however, that just about any company that sells antibodies is likely to have an anti-E. coli poly- or monoclonal available. Some places have a worry free antibody program where you order the antibody and if it doesn't work as you'd like it to, they will refund your money. One I know for sure that does that is Assay Designs. Places like that most likely will also have a good technical staff to be able to guide towards the antibody most likely to work. DH5alpha most likely isn't going to have much difference in its membrane proteins than any other E. coli strain, so that shouldn't be a problem.

#3 peanut142

peanut142

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 12 February 2010 - 10:03 AM

Hey Fishdoc,

Thanks a lot for your reply. I will have a look and update this post if I manage to find a good antibody. Cheers! :)




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.