I think this experiment might be interesting, just to examine whether or not the binding of two different particle, if I were be the one to design, I would first do the dot blot instead of western blot. And for molecular weight analysis, I would use the dot blot result to confirm and then perform native PAGE then western blot.

For dot blot

1. Mark the grid on PVDF (with pencil), as a single grid should contain only a sample.

2. Using a PVDF membrane and activate it using ~100% methanol and balance in PBS/TBS.

3. Apply balanced PVDF on a semi-wet tissue paper to maintain its moisture but keep it wet at the same time.

4. Dissolve different pieces of bacteria in PBS/TBS and load bacteria mixture on the PVDF. Wait until dry completely.

5. Blocking using 5% skim milk of BSA in PBS.

6. Protein staining

7. Wash

8. Antibody staining, you should use monoclonal that only binds to your protein in step 7.

9. Wash

10. ECL

For native PAGE

You should prepare PAGE just the same as SDS-PAGE, only without adding SDS.

Only some notes you should consider:

1. A 7.5% gel would be recommended for unknown proteins. Proteins that has Mw >100,000 should be separated in 3–5% gels. While gels of 5–10% will separate proteins in the range 20,000–150,000, and 10–15% will separate proteins in the range 10,000–80,000.

2. Native protein can be denatured under hot environment, therefore you should perform PAGE in cold room.

3. One can not assure the charge of a native protein, therefore separating gel might need to adjust to an acid condition.

4. Stacking gel may cause native proteins to aggregate, therefore if this will be the problem, just perform the PAGE without stacking gel.

(Protein Protocols Handbook, The, 57–60.)

Best