I am running a Blue Native PAGE (BN-PAGE) and I have been running the gel for 2 hours at 40 V at 4°C and my samples are not running into the gel.  The markers are not running into the gel either, so I know it's not just an issue with the charge/amount of protein loaded.  I'm trying to isolate a complex that is about 500 kDa.  I did not boil my samples or anything, I simply dissolved them in a small amount of detergent (0.5% or 2.5% DDM, or 1% SDS), incubated for an hour on a rocker at 4°C, pelleted out insoluble material, added coomassie brilliant blue G250 to 25% of that of the amount of detergent (from a 5% solution in DI water), and loaded them on the gel.  I developed my protocol from Schagger et al., 1993.

Here's some info on my protocol:

Gel: 4-12% gradient gel (ExpressPlus PAGE Gel; GenScript M41210)

Sample Buffer: 50 mM bis-Tris, 50 mM NaCl, 1 mM EDTA, 5 mM 6-aminocaproic acid, 10% glycerol, pH 7.2

Anode Running Buffer: 50 mM bis-Tris, 50 mM Tricine, pH 6.8

Cathode Running Buffer (First 1/3): 50 mM bis-Tris, 50 mM Tricine, 0.02% Coomassie brilliant blue (CBB) G250, pH 6.8

Light Blue Cathode Buffer: 50 mM bis-Tris, 50 mM Tricine, 0.002% CBB G250, pH 6.8

Molecular Weight Markers: Native Mark (Invitrogen)