Native PAGE to study BiP complexes

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

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

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)

References:

1.     Preissler S, Chambers JE, Crespillo-Casado A, Avezov E, Miranda E, Pérez 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.