Blue Native Gel Electrophoresis

Stock solutions

49.5%T, 3%C Acrylamide
        24 g acrylamide, 0.75 g bisacrylamide / 50 ml H2O
        Store at RT

3 x Gel buffer
        150 mM BisTris-HCl, 1.5 M 6-amino-caproic acid, pH 7.0
        Adjust pH to 7.0 with HCl at 4°C
        Store at 4°C

75% (w/v) Glycerol
        Store at 4°C

10 x Cathode buffer
        0.5 M Tricine, 150 mM BisTris
        No need to adjust pH
        Store at 4°C

5 x Anode buffer
        0.25 M BisTris-HCl, pH 7.0
        Adjust pH to 7.0 with HCl at 4°C
        Store at 4°C

2 x BisTrisACA
        200 mM BisTris-HCl, 1 M 6-amino-caproic acid, pH 7.0
        Adjust pH to 7.0 with HCl at 4°C
        Store at 4°C

50BTH40G
        50 mM BisTris-HCl, 40% (w/v) Glycerol, pH 7.0
        Adjust pH to 7.0 with HCl at 4°C
        Store at 4°C

 

Working solutions

1xCathode (-dye), 1x Anode buffer
        Store at 4°C

1xCathode+dye
        0.01% Serva G in 1xCathode buffer

It takes a while to dissolve ServaG.
Store at 4°C

BN-sample buffer

Serva G

50 mg

2 x BisTrisACA

500 ul

75% Sucrose

400 ul

H2O

100 ul

Store at -20°C

Solubilization buffer

50BTH40G

100 ul

10% Detergent

40 ul

H2O

60 ul

§         Detergent: TX-100, dodecyl-maltoside, digitonin or SDS

§         Water soluble digitonin should be used.

§         If you don’t get water-soluble digitonin, purify as described in Mori et al. (1999) J Cell Biol. 146, 45-55. We've also tried 2X crystalized digitonin with essentially the same results.

§         Note: the original method uses ACA in the solubilization buffer.

5-13.5% gradient blue native gel

Mini gel with 0.75 mm spacer

 

Sample (4%)
for 2 gels

Separation
(5%)

Separation
(13.5%)

49.5% Acrylamide

0.242 ml

0.212 ml

0.573 ml

3 x Gel buffer

1.0 ml

0.7 ml

0.7 ml

75% Glycerol

0

0.14 ml

0.56 ml

H2O

1.72 ml

1.028 ml

0.247 ml

TEMED

6 ul

2 ul

2 ul

5% AP

Pour into a gradient maker

32 ul

11 ul

1.88 ml

11 ul

1.8 ml

Total

3.0 ml

2.1 ml

2.1 ml

Sample preparation

Wash thylakoids with 0.33 M Sorbitol, 50 mM BisTris-HCl, pH 7.0
Resuspend in 25 mM BisTris-HCl, 20% Glycerol, pH 7.0
Adjust [chl] to 2.0 mg/ml (or resuspend thylakoids in the resuspension buffer to 2.0 mg chl/ml)
Add one volume of solubilization buffer, gently vortexing after each drop
Agitate for 30 min (DM, TX-100), 30-60 min (Digitonin) at 0 to 4°C
Spin at ~100,000 g for 30 min
Transfer the supernatant to a new tube
Add 1/10 vol of BN sample buffer
Mix gently
Load 5.5 ul onto BN-gel

 

Protein standard

 

Apoferritin
+BSA

β-amylase
+β-lactoglobulin

5 mg/ml protein stock

7.5 ul each

10.0 ul (amylase)
5.0 ul (lactoglobulin)

Digitonin Solubilization buffer

15.0 ul

15.0 ul

BN-sample buffer

3.0 ul

3.0 ul

Protein stocks in 20% glycerol, 25 mM BisTrsi-HCl, pH7.0

Mix gently
Load 5.5 ul

Protein marker

Mr (kDa)

Ferritin

880, 440

β-amylase

200

BSA

132, 66

β-lactoglobulin

35

Thyroglobulin (670kD) migrates at position close to ferritin dimer (880kD).

 

Electrophoresis

We use a Mighty Small Hoefer setup with a cooling core.
Set a BN gel in a unit connected to a cooling circulator (2-4 C°C in the cathode tank)
Load samples
Run at constant voltage of 100-200 V, 3-6 hrs.

For immunoblotting or fluorography:

Exchange the cathode buffer (+dye) to cathode buffer lacking the dye after top 1/2 ~2/3 of the gel is covered with dye; then run until the free dye has run off of the bottom of the gel (~2 hrs). The gel should look like this:

For convenience, I put the power supply on a timer when I change to dye-minus cathode buffer. The power supply turns off at the end of the run, but the cooling circulator remains on and the gel stays cold until the next morning, when it can be further processed.

 

Immunoblotting

Transfer buffer
        25 mM Tris, 192 mM Glycine, 20% Methanol
        Use room temperature transfer buffer to kill peroxidase-like activities.

Incubate BN-gel in 0.1% SDS, Transfer buffer for ~10 min at RT
        0.5 ml 10% SDS + 50 ml Transfer buffer
Assemble blotting sandwich with PVDF membrane
        PVDF membrane must be wet in methanol before equilibration in transfer buffer
Set in a blotter and run at constant voltage of 50 V for 45 min. - no ice in the chamber.
Stain the blot with 0.1% PonceauS, 5% acetic acid
        It’s a little hard to see marker proteins.
Destain with 20% MeOH, 7% acetic acid overnight to improve antibody binding. (I've been skipping any staining and destaining steps; KC.)

Treat the blot like ordinary immunoblotting

 

Fluorography

Treat with DMSO and then PPO/DMSO like an SDS-gel.
(It's a little hard to see the MW stds of gels treated this way; I can see the orange ferritin bands and BSA monomer. Hiroki stains and detains his gels before PPO/DMSO to see the markers. I don't stain in order to avoid color quenching; KC)

 

2-Dimensional Analysis

The BN-gel is run as described above on a 0.75 mm thick gel. The lane of interest is sliced out with a razor blade and then incubated in SDS sample buffer containing 2.5% mercaptoethanol for 10 min. at room temp. or 25°C. The slice is then placed on top of a 1 mm SDS-PAGE gel. We use a BioRad minigel apparatus for this. The stacking gel is poured with a comb that is mostly flat across but has a tooth on one side for mol. weight markers, etc. We've tried sealing with agarose or acrylamide, but it seems to work fine without any sealant. The 0.75 mm gel slice slides nicely onto the 1 mm stacking gel without a lot of pushing and tearing. We always use fresh gel slices (no freezing). Here is a 2D gel done by Lixin Zhang:

Good luck

Hiroki Mori at 7/12/2000