Native PAGE to study BiP complexes
[This method was prepared by Steffen Preissler and used in two recent papers: (1,2); posted 6 May 2016]
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
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
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 before removing the comb. Gels can be wrapped in tissues soaked with running buffer and stored @ 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 lysate for subsequent Western blotting. Always use fresh samples and load them as soon as possible. Spin samples 1 minute @ 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.
- Equilibrate PVDF membrane 2-5 minutes in MeOH & rinse thoroughly with transfer buffer
- Incubate the gel in transfer buffer for at least 5 minutes before assembly of the blot
- Assemble the blot with 1 x Bjerrum transfer buffer containing 0.04% SDS
- Blot for 1 h @ 100 V with ice in the tank or preferentially 16 h @ 30 V (add stir bar to mix overnight)
- Wash membrane 20 minutes 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)
1. Preissler S, Chambers JE, Crespillo-Casado A, Avezov E, Miranda E, PŽrez J, et al. Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker. elife. 2015;4.
2. Preissler S, Rato C, Chen R, Antrobus R, Ding S, Fearnley IM, et al. AMPylation matches BiP activity to client protein load in the endoplasmic reticulum. elife. 2015;4.