Fractination of SA into Cytosol., Cell Wall, Cytoplasmic

[RuNkLi]

Hi,

i have to seperate protein fractions of cytosolic proteins, cell wall proteins and cytoplasmic proteins. the reason is, i want to find out where the antimicrobial peptide is located after incubation with S. aureus.

I just control with Western Blot.

The problem is, i can't find a protocol for isolation or digestion of these fractions. We have no ultracentrifuge!

I would be very happy for helping...

Best regards

Ilknur

[blcr11]

So the peptide is labeled with something like P-32 and you're trying to see whether the label is retained with the membranes or is floating in the cytoplasm? I don't know how you're going to make sure the peptide stays bound to whatever it's bound to unless you cross-link it. That might work for something bound to the surface, but it shouldn't touch anything bound to something cytosolic. And then, if you're checking things out by Western, I guess you have an anti-peptide Ab with an appropriate Anti-Ab conjugate for detection, or something like that. But does the peptide stay bound under the conditions of SDS-PAGE? or do you care once the partition has been made? I don't know, maybe I'm just not thinking about this correctly, but it sounds tricky to me.

I can't see how you're going to do this without any centrifuge, but perhaps you don't need a full-blown ultracentrifuge. If you have access to either a benchtop Eppendorf centrifuge, which can spin a 1.5 ml tube up to 17,000xg, or an RC-5Plus or equivalent which can spin 15-30 ml corex tubes at something like 17-20,000xg, that should be enough.

Any standard extraction (freeze-thaw or french press or sonication) will get you a separation between soluble and probably cytoplasmic versus insoluble probably in inclusion bodies or membrane bound. You can also try something like BugBuster from Novagen (now EMD Biosciences) that doesn't require any additional disruption--but you do have to spin down at least twice--an RC-5 Plus is good enough for this. You can also fractionate soluble protein from insoluble protein by vacuum filtration--but I wonder if your peptide will be bound strongly enough to partition correctly in the presence of something like BugBuster reagent (essentially a cocktail of detergents).

Don't know if that helped or not. Good luck with it, anyway.

[RuNkLi]

First thank you very much for your detailed answer.

In immune electronmicroscopy pictures we can see, that this peptide binds to surfacial proteins as well as cytoplasmic structures?. We want to confirm, make sure and find the mode of action by finding out these targets.

The point you mention is very correct. I am not sure if this binding of peptide to different structures is really strong enough that i can do this very dirty experiments.

We have just produced AB against this peptide. They are not radioactive labeled. I can only detect them by Western blot. Another way is may detect them by FACS because we have FITC labeled peptides. But I'm not sure if I can do this by this way

I will try freeze-thaw protocol, may this is the softer way for breaking the protoplasts.

I just want to try may I have luck. Who knows

Thanx

IS

[Ewa]

1. extracellular fraction - secretory proteins - spin down the culture, precipitate

2. cell wall fraction - wash the cells from (1) thoroughly, suspend in raffinose (or sucrose - it is even cheaper), lyse with lysostaphin, collect the supernatant after spinning down

3. cellular + membrane fraction - use a detergent/extensive pipetting/freeze-thaw or just osmotic shock, whichever you prefer
- spin down (at ultra speed) to collect rest of membranes, supernatant is soluble cytosolic fraction plus some membrane fraction contamination

my hint - DO use protease inhibitors throughout the procedure

my own small scale lab protocol for staph fractionation is as follows:
1. grow the strain in an appropriate medium (TSB or BHI) at 37C/200 rpm till stationary phase (overnight) and collect the culture samples
2. spin down the cells (5-6K rpm/ up to 30 min/4C)
3. collect the supernatant containing extracellular proteins, you may need to concentrate them (AmSO4/memebrane ultrafiltration: vivaspin, centricon, whatever else 10 K cut-off) and store at 4C
4. wash the cells thoroughly with fresh media /PBS/whatever you have (add 1-5 ml, resusupend cerafully and spin down). Repeat this step to be sure that you’ve removed all the reamainings of secretory proteins
5. resuspend the cells in 50 mM Tris-HCl pH 7.5, 20 mM MgCl2, 30% raffinose (Sigma, recommended, but ANY sucrose does the trick as well)
6. lyse the cells with lysostaphin [our stock solution is 5 mg/ml, working concentration 0.1 mg/ml]
7. incubate at 37C for 30 min, suspension should not become sticky, if it does it means the cells have lysed and the cell wall fraction will be contaminated with cytoplasmic proteins
8. remove the protoplasts by centrifugation (5-6K rpm/30 min/4C) and collect the supernatant containing the cell wall-associated proteins
9. do NOT freeze the wall fraction! Keep it at 4C. you can try to dialyse it if you’re doing a larger scale preparation
10. keep the protoplasts on ice at all times, be careful with pipetting them as they are really fragile, cut the top of the gilson’s tip off
11. wash the protoplasts twice with ice-cold 50 mM Tris-HCl pH 7.5, 20 mM MgCl2, 30% raffinose, spin down (4-5K rpm/15 min/4C) and discard the supernatant.
12. optional: add inhibitor mix
13. lyse on ice by adding in an ice-cold 50 mM Tris-HCl pH 7.5, 20 mM MgCl2 and by repeated vortexing and pipetting.
14. optional: add 0.5 - 1 ul of DNase if the sample is really sticky
15. centrifuge the lysate (max/30 min/4C), collect the supernatant
16. run the samples on SDS-PAGE gels, no need to load more than 1-5 ul of the cytoplasmic fracton as they are usually concentrated

LYSOSTAPHIN (Sigma cat no L-7386), a glycyl-glycine endopeptidase from Staph. staphylolyticus that cleaves pentaglycine bridges crosslinking the peptidoglycan in the cell wall - store at -20C.

DNase (Roche) 10 units/ul

Complete EDTA-free Protease Inhibitor Cocktail, (Roche, cat no 1 873 580), you can make one on your own using E-64, DIC and O-phenentroline