You may try tagging the protein with a fluorescent protein like GFP, expressing the tagged protein in the bacteria and observing the localization through a good enough (super-high resolution) confocal microscope. Go for C-terminal tagging as the N-terminal usually contains signal peptides important for localization.

Dear Chenhao li

if you have antibodies able to bind the protein in the folded state you can try label it with a fluorophore and to perform FACS orhigh resolution confocal microscopy

(Single cell super-resolution imaging of E. coli OmpR during environmental stress - Integrative Biology (RSC Publishing) DOI:10.1039/C5IB00077G)

a simple, but more complex and less reilable possibility is perform a selective membrane extraction and check the presence of the protein in the fraction using the WB.

In absence of any antibody, surfome analisys throgh mass spectrometry, may be an alternative way:

https://www.nature.com/articles/s41598-019-53493-8

best regards

Manuele

Thanks for your suggestions. Manuele Martinelli Ananth Krishna Narayanan

Hi Chenhao,

You can try observing with a super-resolution microscope.

https://www.creative-biostructure.com/Super-resolution-Microscopy-Service-590.htm

Dear Chenhao li . See the following useful RG link: Article Protein Subcellular Localization in Bacteria

Kindly see also the following useful RG link:,Article Evidence that subcellular localization of a bacterial membra...

Also check please the following useful RG link: Article Predicting subcellular localization of multisite proteins us...

The following RG links are also very useful: Article Systematic Localization of Escherichia coli Membrane Proteins

Article Protein Localization in Escherichia coli Cells: Comparison o...

https://www.sciencedirect.com/topics/medicine-and-dentistry/protein-localization

Visit also the following useful RG links: Article An Improved Method for Bacterial Immunofluorescence Staining...

https://www.frontiersin.org/articles/10.3389/fchem.2020.603259/full

Thanks for all your help! very useful information!@ Traci L Testerman Aref Wazwaz Roger Davis Manuele Martinelli Ananth Krishna Narayanan

You are most welcome dear Chenhao li . Wish you the best always

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845201/,kindly check this link.

Often, polarly localized proteins are recruited to the poles through their interaction with other proteins or protein complexes that were already located there, in a so-called diffusion-and-capture mechanism.Jan 1, 2014

https://pubmed.ncbi.nlm.nih.gov › ...

How do bacteria localize proteins to the cell pole? - PubMed

Typically, isolation of recombinant proteins from bacteria involves a cascade of operations, including cell harvest, cell disruption, and homogenate clarification to remove cell debris, followed by a combination of chromatographic methods to obtain highly pure protein (Demain and Vaishnav, 2009).Aug 10, 2015

https://www.sciencedirect.com › pii

A microscale method of protein extraction from bacteria: ...

The most widely used method for protein purification is affinity chromatography, which separates proteins based on their specific interaction with a matrix. It is one of the most effective techniques, since it takes advantage of the incorporation of a structure of choice (called a tag) onto the protein.Feb 11, 2014

https://www.sepmag.eu › blog › bid

Recombinant protein purification: The 6 most effective methods

https://www.biorxiv.org/content/10.1101/855171v1.full,kindly check this link.