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Blue native PAGE
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Blue native PAGE

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Category
Isolation & structural analysis of glycans
Protocol Name

Blue native PAGE

Authors
Harada, Yoichiro
Glycometabolome Team, RIKEN Advanced Science Institute
KeyWords
Reagents

Reagents required for BN-PAGE are commercially available from Life Technologies, Carlsbad, CA (Bis-Tris/tricine buffer system).

To prepare the reagents in your Lab, please see below (Immidazole/tricine buffer system). All the procedures are based on the previous report [1].

1 M imidazole/HCl, pH 7.0, store at 4ºC.

1 M imidazole (non-pH-adjusted stock), store at 4ºC.

10% (w/v) digitonin (Calbiochem, San Diego, CA) (see Note 1).

1 M tricine, store at 4ºC.

2 M 6-aminohexanoic acid, store at 4ºC.

Acrylamide-bisacrylamide mix (AB mix; 49.5% T and 3% C)(see Note 2), store at 4ºC.

3×Gel buffer (75 mM imidazole/HCl, pH 7.0 and 1.5 M 6-aminohexanoic acid)(see Note 3), store at 4ºC.

10% (w/v) ammoniumpersulfate (APS), store at 4ºC.

5% (w/v) Coomassie Brilliant blue G-250 in 500 mM 6-aminohexanoicacid, store at room temperature.

Ponceau S/glycerol stock (0.1% Ponceau S and 50% glycerol, w/v), store at 4ºC.

Cathode buffer + Dye (50 mM tricine, 7.5 mM imidazole (non-pH adjusted), 0.02% Coomasie blue G-250), prepare when use and chill at 4ºC.

Cathode buffer + Dye/10 (50 mM tricine, 7.5 mM imidazole (non-pH adjusted), 0.002% Coomassie blue G-250), prepare when use and chill at 4ºC.

Anode buffer (25 mM imidazole, pH 7.0), prepare when use and chill at 4ºC.

High molecular weight native marker kit (GE Healthcare, Little Chalfont, UK)

Instruments

Conventional apparatuses for polyacrylamide gel electrophoresis (see Note 3)

Gradient mixer

Peristaltic pump

Methods
1.

Preparation of native polyacrylamide gel

1) 

 Mix the reagents on ice (CRITICAL POINT!) listed in Table 1, except for APS and TEMED.

Table 1 Compositions of the stacking gel and the gradient separating gel for a mini-gel.

Reagents

Stacking (3.5%)

Separating (4%)

Separating (13%)

AB mix

0.44 mL

1.5 mL

3.9 mL

3x Gel buffer

2 mL

6 mL

5 mL

Glycerol

0 mL

0 mL

3 g (2.4 mL)

Water

3.4 mL

10.4 mL

3 mL

10% APS

50 μL

100 μL

75 μL

TEMED

5 μL

10 μL

7.5 μL

Total volume

5.895 mL

18.010 mL

14.3825 mL

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2) 

 Set up the gradient mixer and peristaltic pump (Fig. 1A). Put a small stir bar into the chamber A.

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3) 

 Add APS and TEMED to the 4% separating gel solution and pour 3.75 mL of the solution into the chamber A.

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4) 

 Fill the connecting tube with the solution.

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5) 

 Slant the gradient mixer to lean the solution to the camber A.

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6) 

 Close the cock.

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7) 

 Add APS and TEMED to the 13% separating gel solution and pour 3.50 mL of the solution into the chamber B.

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8) 

 Start stirring and pumping.

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9) 

 Open the cock on the connecting tube.

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10) 

 Open the cock between the chamber A and peristaltic pump.

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11) 

 Pour the gradient solution into the gel plates.

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12) 

 When all the solutions get into the gel plates, overlay a small volume of water on top of the gradient solution.

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13) 

 When polymerized, pour the stacking solution that has been mixed with APS and TEMED.

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14) 

 Insert a comb and wait until polymerized.

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2.

Sample preparation for BN-PAGE

1) 

 Prepare samples of interest in a 10–50 mM buffer, pH 7.0–7.5 containing NaCl less than 50 mM. Add non-ionic detergents, if necessary.

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2) 

 Centrifuge at 15,000 × g for 5 min and recover the supernatant.

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3) 

 If the sample is purified, add one tenth of volume of Ponceau S/glycerol stock to the sample just before loading to gel. If the sample is crude, add one fifth of volume of 50% glycerol and one twelfth of volume of 5% Coomassie G250 to the sample just before loading to gel. Add non-ionic detergents at 1%, if necessary.

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3.

BN-PAGE

1) 

 Set the BN-gel to the PAGE apparatus.

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2) 

 Pour the cathode buffer + Dye into the inner chamber.

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3) 

 Pour the anode buffer into the outer chamber.

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4) 

 Load the sample into the well. If it will be difficult to see the well, use a backlight.

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5) 

 Run the gel in a refrigerator or in the cold room with a constant voltage at 100 V.

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6) 

 When the samples completely enter the gel, change the setting to the constant current at 15 mA until the dye frond reaches at approximately one third from the top of the gel.

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7) 

 Aspirate the cathode buffer + Dye completely and pour the cathode buffer +Dye/10.

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8) 

 Stop the electrophoresis when the dye front reaches at the bottom of the gel.

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4.

Staining

1) 

 If the amounts of the sample are sufficient, you will see the protein bands immediately after the electrophoresis.

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2) 

 If you need to stain the gel with silver, destain the gels over night in methanol/acetate/water (5:1:4, v/v/v) and then perform the silver staining (Fig. 1B).

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3) 

 For Western blot, perform the regular electroblotting with PVDF membrane (Do NOT use nitrocellulose membrane!). After the electroblotting, wash the membrane three times with water and destain with methanol. After the destaining, wash the membrane three times with PBST and proceed to Western blot. Depending on antibodies, they may not bind to the antigens that retain their native conformation or are embedded in the structural part of the proteins. In these cases, the antibodies may recognize the antigens after the membrane was denatured for 30 min at 50ºC in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 0.8% β-mercaptoethanol.

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Notes

1. Aliquot in a small volume and store at −80ºC.

2. ‘% T’ indicates the total concentration of acrylamide and bisacryamide. ‘% C’ indicates the percentage of bisacrylamide. Mix 48 g acrylamide and 1.5 g bisacrylamide in 100 mL of water.

3. Detergents have to be completely washed out.

Figure & Legends

Figure & Legends

 

 

Fig. 1. BN-PAGE

  (A) Preparation of a native polyacrylamide gel. (B) An example of the separation of the molecular weight marker by BN-PAGE, followed by the silver staining. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and albumin (66 kDa). 

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