Hi Sameh,

This will depend if you want to extract a protein that you are overexpressing with a tag or if you just want to extract the bacterial protein from a stress condition where you know it expresses the heat shock protein. If you don't have the gene cloned and you only want to extract the protein. My advice is to grow the bacterial under your stress condition and lysate it with SDS 0.1 % and do and SDS-PAGE. If you know the weight of the protein you can cut it from the gel. But if you want it pure and in more ammount, you need to clone the gen in a expression vector and purified depending on the tag you use in the vector. 

Hopefully this can help you

Claudia

many thanks Dr Claudia. Can I ask you for an official protocol of the method you mentioned, SDS 0.1%. many thanks again

the method mentioned by Cloudia is correct. another way to extract protein from microbial cells is by sonication, if it is intracellular. After sonication, centrifuge the suspension at 3000 rpm to separate debris. If you want to concentrate the protein, mix acetone gradually to it (double of the volume of the protein extract).Keep in the fridge over night. Next day cloud of proteins will appear which can be separated by again centrifugation. Check the protein concentration by any standard method.

Hi Sameh 

you need to grow the bacteria and pellet by centrifugation

sorry it did not upload complete.

After centrifugation wash the pellet with PBS, and then resuspend the pellet in 0.1% SDS in PBS, vortex, measure the protein concentration and use the lysate to load the gel.

Claudia

Hi Claudia,

Would your protocol achieve high yield on both Gram-negative and Gram-positive bacteria? I'm wondering if SDS alone is enough to lyse the peptidoglycan layer. I need to extract a low abundance protein into a low volume of buffer (to achieve higher concentration) so I'd like to avoid sonication as best as I can. 

Hi Sameh,

If your protein MW is not close to 18 kDa, you can also add lysozyme to your lysate to increase lysis efficiency. Another way is to use commercial buffers available (B-Per, BugBuster,...) if you have to process a large number of samples in a short time. 

Hi Thu,

I used this protocol for E. coli and C. difficile and it worked fine, but I only used it to see the protein pattern and to do western blot not for obtaining the protein. If you want to lysate Gram positives you can use lysozyme and proteinase K. Hope this works for you.

Thanks

Claudia

Hi all,

is there any protocol in pdf file, or a link of a publication, including a protein exctraction of bacteria?

Efthymios Poulios here is a recent paper, protein isolation using Trizol: Article Proteomics of intracellular Salmonella enterica reveals role...

Found this one as an alternative too, but I haven't tried any of these: http://2016.igem.org/wiki/images/e/ef/T--Wageningen_UR--Protein_Extraction.pdf

Thu Tran why did you want to avoid sonication? what's the disadvantage of it?

I want to do proteomics on Salmonella (G-), but I don't obtain enough proteins using RIPA buffer, it may be mild for Salmonella. I wonder whether there is an isolation protocol without Trizol.

Thanks,

- Hande

Thanks a lot. Best regards.

Claudia Fabiola Martinez de la Peña, what will be the centrifugation speed. And PBS concentration.?