Hi Pedro J. Hernando

Just to clarify - it sounds like you can successfully amplify the lecA gene from your PAO1 cultures by PCR. You then grow the strain, collect some of the culture, lyse in SDS sample buffer, and attempt an anti-LecA Western Blot with whole cell lysate, but fail to detect LecA protein. You use a recombinant/purified LecA protein as a control, and this can be detected in a control Western blot, suggesting that the antibody is not the problem. Am I understanding this correctly?

Some suggestions:

1. LecA may only be expressed under very specific conditions. I recommend reviewing the literature and checking to see if anyone has ever reported the conditions for expression. Things to consider are growth phase (early exponential, late exponential, stationary?), temperature, media type, etc. LB broth is infamous for being a very poor choice for physiological studies: researchers use it because it is cheap and bacteria grow rapidly in it, but has major limitations for these types of studies.

2. You report 10^8 cells, but is this your entire extract that is loaded? Have you checked total protein content, and normalized your loadings? Typically antibodies have a low limit of detection, but if the LecA protein is not very abundant and your loadings are on the low side, you may fall below the detection limit. I recommend lysing cultures and measuring total protein by Bradford or BCA (make sure your lysis buffer/matrix is compatible!). If I recall correctly, for best results 10-well SDS-PAGE gels are generally loaded with 1-10 micrograms of total protein, but if LecA is not very abundant you may need to load much more (50-100 ug).

3. What is your primary antibody? That is, are you blotting against epitope-tagged LecA, or is the antibody a monoclonal/polyclonal raised against native LecA? In your control experiment, are you using a recombinant LecA from PAO1, and is it denatured and run on an SDS-PAGE, then transferred to a membrane and blotted? Small differences in amino acid sequence from closely related LecA homologs or an antibody that recognizes a noncontinuous epitope could be creating problems for you.

4. What is your Western detection method? Some secondary antibodies are much more sensitive than others - consider an HRP conjugate for detection if you are using AP currently.

Best of luck with the experiment!

ACA

In how many isolates have you been able to cultivate...mayb the quatity is low...increase growth of the identified isolates and ..plus what methods are you following.

P. aeruginosa produces two small soluble lectins, LecA and LecB (also named PAI-L and PAII-L, respectively) that interact with specific sugars. Crystal structures have been solved for both, and binding affinity experiments showed that LecA binds to galactose and its derivatives, while LecB binds to fucose, mannose, and mannose-containing oligosaccharides9,10,11,12. In addition, it is important to note that functional LecB is a homotetramer consisting of four 114 amino acid LecB monomers which require two divalent calcium ions and has been shown to be associated with the outer membrane. Ref below

https://www.nature.com/articles/s41467-019-10201-4