Native-PAGE to study BiP oligomeric complexes

Refer to Bio Rad Mini-Protean gel system and handbooks to read about PAGE basics and principles.

Tris-glycine gel system with 4.5 % stacking gel and 7.5% separation gel. Stacking and separation gel are both Tris-buffered with pH 8.8.

3x loading dye:

240 mM       Tris-HCl pH 6.8

30%              glycerol

0.03%           Bromophenol blue (from 0.4% w/v stock in ddH₂O)

                     See Thompson AD et al., 2012, Cell stress chaperones 17:313-327

10 x Running buffer (500 ml): 

15 g        Tris-base

72 g        glycine

Intended pH 8.3-8.9 (do not adjust)

Stacking gel (4.5%)

Acrylamide [T = 30%/C = 2.6%]         1.5 ml

1.5 M Tris-HCl pH 8.8                         0.8 ml

ddH₂O                                                7.6 ml

10% APS                                             100 µl

TEMED                                                10 µl

Separation gel (7.5%)

Acrylamide [T = 30%/C = 2.6%]         10 ml

1.5 M Tris-HCl pH 8.8                         10 ml

ddH₂O                                                19.6 ml

10% APS                                             400 µl

TEMED                                                32 µl

Pour the gels freshly before the run. Use 0.75 mm spacer plates (preferable standard) but 1 mm or 1.5 mm spacer might be more suitable for native-PAGE with lysates. Polymerize stacking gel for 1 hour in horizontal position (lay gel stand flat to avoid collapse of wells) before removing the comb. Gels can be wrapped in tissues soaked with running buffer and stored air-tight @ 4°C for up to five days. Wash the wells before loading. Optional: pre-run the gel until current stays constant. Load 5 µg purified protein per lane for Coomassie staining. Load 30-50 µg mammalian cell lysate for subsequent Western blotting. Always use fresh samples and load them as soon as possible. Spin samples 1 min @ full-speed in a microcentrifuge immediately before PAGE. Run gel 2 h at constant 120 V. Optional: After the run rinse the gel briefly in ddH₂O and stain or proceed with Western blotting.

Western blotting of native gels

Use Bjerrum transfer buffer without MeOH with 0.04% SDS for transfer of native proteins to a membrane. Use Bio-Rad wet-blot transfer system compatible with Mini-Protean gels. Transfer to PVDF membrane. See also transfer buffer guidelines from Roth. Transfer buffer conditions (MeOH and SDS content) should be optimized for each protein of interest.

1. Equilibrate PVDF membrane 2-5 minutes in MeOH & rinse thoroughly with transfer buffer

2. Incubate the gel in transfer buffer for at least 5 minutes before assembly of the blot

3. Assemble the blot with 1 x Bjerrum transfer buffer containing 0.04% SDS

4. Blot for 1 h @ 100 V with cooling block in the tank or preferentially 16 h @ 30 V (add stir bar to mix overnight @ RT)

5. Wash membrane 20 min with 1 x Bjerrum transfer buffer containing 20% MeOH to remove SDS & block the membrane in absence of any detergent

10 x Bjerrum transfer buffer (500 ml)

29.1 g Tris-base

14.65 g glycine

pH 9.2 (adjust if necessary with Tris or glycine)

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