hello,
Iam not expert in bacterilogy, iam looking for specific surface antibody against this particular E.coli strain (MG 1655),it expresses GFP? .my aim is to bind/adhere the whole bacteria on glass slide
i used this particular antibody (pls check the attached file) ,but with no success, i dont know if this antibody is good enough for MG1655 E.coli
help will be highly appreciated!
thanks
Why do you need to use an antibody for the adhesion? You can just use poly-lysine, as it binds LPS (surface lipopolysaccharide) and immobilizes cells. Poly-lysine is commonly used for slide preparation for microscopy. If you really need antibodies gainst E. coli surface, you can try anti-LPS antibodies or those raised against whole cells of E. coli.
thanks for your reply, but my goal is to have intact LPS on e.coli, which i will use fo ractivation of other cells so i dont want to use LPS as antigen to bind, i want some other surface antigen which can bidn e.coli on glass and keep its LPS activity intact,can you suggest some antibodies raised agaisnt whole e.coli (MG 1655 strain)
Antibodies raised against whole cells will also react with LPS. If you want to avoid it, your best bet is anti-OmpA and anti-OmpC antibodies. OmpA is the major outer membrane protein, which has long extracellular loops and functions as an adhesin. Another outer membrane protein to try is OmpC, but I would recommend starting with OmpA.
Here are commercial antibodies (I have not used them, so I can't tell you how good they are).
OmpA: http://www.antibodyresearch.com/ompa-arc111120.html
OmpC: https://www.linscottsdirectory.com/antibodies/lifespan-biosciences-inc/Outer-Membrane-Protein-C-14405538
thank you very much,it is god help for me, i will try to get the Omp A antibody
do u think i can get biotin conjugated one of this antibody?
another question is ,the antibody that i have already (attached ) is that good for MG1655 irrespective of LPS?
It is likely that the antibodies, that you already have, were raised from total cell lysate, and there will recognise different proteins, mostly cytoplasmic. It's not stated in the description, but I assume so because the purpose of antibodies is to remove E. coli protein from the protein preparations. I don't know whether these antibodies would recognize OmpA or any other surface proteins. It has to be tested.
As for OmpA antibodies, I can't find a biotinylated version, but it is easy to do it yourself, many companies sell kits for biotinylation. There are biotin-conjugated antibodies for OmpC sold by the same company I told you above.
ok i will check that , Thank you very much for info. and suggestions!! it was a great help indeed!!
You are welcome! By the way, if you are planning with use biotin tag for immobilization, you can also biotinylate E. coli cells as well!
thanks , but this biotinylation of E.coli by (NHS sulfo Biotin) could effect the LPS activity
isnt it?
NHS reacts only with primary amines (e.g. Lysine side groups). LPS does not these groups, so it will not be labeled.
Sorry to ask you again, do you think using Poly L Lysine for surface binding of E.coli (K12, MG1655) will effect its membrane function and whole cell viability?
Badri, Poly-Lysine bind LPS and it will immobilize E. coli on the surface. Because of its cationic nature, it will increase outer membrane permeability, and it is toxic, cells will eventually die (not immediately). Despite its drawback, it is often the best thing for microscopy where agarose slides give too much background. Depending on what you need to do, you may still get away with Poly-Lysine.
thank you for the reply, i wonder what if one uses very low concentration of PLL ,say 0.001% or less than that, if that increase cell viability and doesnt harm cell very much for long time
I think this is something that you can test, there is a live/dead staining for bacteria if you want to monitor viability carefully.
Hi, i have one quiry regarding PLL (i am trying to use this untill antibody comes)
if i use PLL to coat GFP E.coli strain, to chekc the viab ility of e.coli ,can i assume cell which are fluorecent GFP as viable cell? this strain has the GFP plasmid vector
No, GFP is a very stable protein and will give you fluorescence even when cells are dead, as long as they did not lyse and release their cytoplasmic content. You can try propidium iodide, live cells should not stain.
ok I see, didnt know that, can i use Trypan blue instead of propium iodide ?
I am not sure. In principle, Trypan blue should not penetrate live cells, but since Polylysine permeabilizes membranes, so it may. I guess propidium iodide would have same problem. Dye which depend on membrane potential are your best bet.
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