SDS-PAGE to resolve free monomeric Ubiquitin (MW 8.6kDa) and subsequent western blot detection.

 

Laemmli SDS-PAGE system has insufficient resolving power below 10kDa. Need to use modified protocol developed by Schagger and Von Jagow (1987), Anal. Biochem. 166, 368-379.

 

„          Following cell lysis in appropriate lysis buffer (± inhibitors of de-ubiquitinating enzymes, where required) protein assay the lysates.

 

„          Mix desired quantity of lysate with SDS-PAGE sample buffer (containing FRESH 100mM DTT). 2-mercaptethanol-based 5x sample buffer seemed to cause lots of problems on the resultant blots (for unknown reason(s)?). You can detect free ubiquitin nicely in just 5mg total lysate protein.

 

„           Preparation of Gels:

 

            Pour 15% (total) acrylamide gels, cast with 30% Protogel but containing double the usual amount of bisacrylamide cross-linker (19:1 ratio) Š NOT the typical 37.5:1 ratio solution.

 

            Gives smaller pore size, hence better separation of low MW peptides.

 

            Better to pour 0.75mm thickness gels to increase transfer of smaller peptides like ubiquitin.

 

„          Running of Gels:

 

Anode buffer (external):      usual Laemmli Tris-Glycine-based running buffer

 

Cathode buffer (internal):   Tris-Tricine-based running buffer

(0.1% SDS; 0.1M Tris; 0.1M Tricine) Š pH doesnÕt need adjusting (should be approx. 8.25)

 

It may be better to run the gels at a lower voltage (say, 80-100V), for longer, rather than the usual 125V.

 

„          Soak gels in a reducing buffer containing 2.3% SDS, 5% 2-mercaptoethanol. 63mM Tris-HCl (pH 6.8) for 30 minutes at RT.

 

„          Electro-transfer of resolved proteins:

 

            Use 25mM 3-[Cyclohexylamino]-1-propane-sulfonic acid (CAPS; Sigma #C6070) buffer (pH 10) with 20% v/v methanol.

 

            Transfer in Genie blotter for at least 30 minutes (donÕt really need to do it for 1 hr though)

 

            Transfer to 0.2mm pore-size membrane (NC or PVDF) to prevent the ubiquitin passing through the membrane. NC gives cleaner blots.

 

„          Heat-inactivation:

 

            Prior to blocking, boil the membrane for 30 minutes in deionised water. This is believed to expose hidden antigenic sites on the ubiquitin molecule (Swerdlow, P. et al. (1986) Anal. Biochem. 156, 147-153).

 

„          Block membrane in 0.5% Cold Water Fish Gelatin (Sigma #G7765), 0.1% Tween 20 in PBS for 2hrs, or overnight at 4oC. Antibody incubations are also in this buffer.

 

„          Use Sigma anti-Ubiquitin (#U5379) antibody as primary, at 1:1000, for 1.5 hrs at RT.

 

„          Wash 6x 5mins in blocking buffer. Add anti-rabbit-HRP secondary (1:2000) in blocking buffer for 1 h at RT.

 

„          Wash 6x 5mins in blocking buffer again, then twice in PBS alone. Perform ECL and develop.