Goal: Disulphide bridges detection by AMS alkylation.
References: Rodríguez, I. et al, 2006 (PMID: 16537584) with modifications.
Procedure: AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, Molecular Probes) alkylates free cysteines, adding 0.5 kDa. Therefore, alkylation of reduced and non-reduced sample led to different electrophoretic size.
1. You need four different samples:
i) Non treated ii) Reduced (this control is essential since reduced protein may change mobility). iii) Alkylated (only free cys will shift electrophoretic mobility). iv) Reduced and alkylated (all cys bounded or not will shift electrophoretic mobility).
Eventually you may prepared a oxidize control, pre-treating the sample with 20 mM H2O2.
This protocol can be used for bacteria or eukaryotic cells. For reducing samples, add 200 mM DTT and let the culture go on for 30 min extra.
2. Extracts preparation:
-Wash the cells with ice cold PBS.
-Precipitate intact cell proteins with cold 10% TCA. You may add 100% TCA or 15% TCA in corresponding amount.
-Incubate 30' at 4ºC
-Centrifugate 15' at 14k rpm
-Wash with 0.2 mL of ice cold acetone and centrifuge again
-Let the pellets air dry for 30' at 25-30ºC
-Resuspend in 200 mM Tris/HCl pH 8, 3% SDS with or without 15 mM AMS
-Incubate overnight at 4ºC
- Alternatively, you can avoid the TCA precipitation by lysis the cells directly in GuHCl 6 M (in 50 mM Tri/HCl pH 8) with or without 15 mM AMS
Optional: remove the non incorporated AMS by a G25 Sephadex gel filtration.
-Use Laemmli loading buffer without any reducing agents.
-For proteins smaller than 25 kDa a Tricine Gel is required, whereas for bigger proteins standard 15% acrylamide glicine SDS-PAGE may be ok for difference the bands.
Then visualize the bands by Coomasie staining or Western blotting.
Stoned pellet: boil the sample in the presence of 5 uL extra Trizma base (no pH adjusted)