Native Gels with Bis-Tris

Stocks/Solutions (Store at 4 \xB0C)

1. 10% w/v Dodecyl b-D Maltoside

2. 20% w/v Digitonin (Fluka #37006)

3. 10% w/v Sodium deoxycholate (DOC)

4. 10% v/v Triton X-100

5. 5% Coomassie BBG250 (in 500mM 6-Aminohexanoic acid)

6. 0.1% Ponceau S in 50% Glycerol

7. Acrylamide (49.5%T/1.5%C)

8. 10% Ammonium Persulfate (APS), Make fresh from a desiccator stored powder.

9. 10X Cathode Buffer: 0.5M Tricine (8.95%)/ 150mM Bis-Tris (3.2%).

10. 10X Anode Buffer: 0.5M Bis-Tris/HCl (10.46%) (pH 7.0)

11. Gel/Sample Buffer (4X): 2M 6-Aminohexanoic acid (26.24%)/ 200mM Bis-Tris (pH 7.0).

Casting the 4.5-13% acrylamide gradient gel

\xB7 13% acrylamide solution has 20% glycerol.

\xB7 Keep the APS and TEMED at 50% of their normal volumes in the casting mix.

\xB7 The gradient maker and acrylamide solutions should be chilled prior to use.

\xB7 Pour the 4.5% mix in to the well of the gradient maker that is away from the out-let, while keeping the valve between the two wells closed.

\xB7 Open the valve briefly to let a small volume of this mix pass through the connecting bridge, so that any trapped air bubbles are flushed out.

\xB7 Add 13% mix to the other well (the one which has the outlet). Switch ON the stirring bar, and start the pump connected to the outlet.

\xB7 Open the valve connecting the two wells of the gradient maker.

\xB7 After casting the gradient carefully over-lay it with the gel buffer (1X).

\xB7 It takes about 1H for the gel to polymerize.

\xB7 Don't forget to flush the gradient maker and its tubing, with water, to avoid polymerized acrylamide choking it!

Stacking Gel:

\xB7 Stacking gel has 4% acrylamide and the mix need not be chilled. After it is polymerized (in about 20min) the comb is removed and wells are washed and filled with the gel buffer (1X).

\xB7 Store the gel at 4 o C .

Sample preparation

\xB7 5ul of “dry” mitochondrial pellet is solubilized, for 15-20 min on ice, in 20ul (final volume) sample buffer with 2.5% Dodecyl maltoside.

\xB7 Solubilized sample is centrifuged at 25000xg for 20 min at 4\xB0C.

\xB7 Collect the supernatant and estimate the protein.

\xB7 Add 1ul of 5% CBBG or 0.1% Ponceau for 10ul of sample before loading .

Running Conditions:

\xB7 Run in a cold-room.

\xB7 Samples are run at 75V in a mini-gel apparatus for up to 2h depending on the detergents used in the cathode buffer.

Histochemical Assays (in10ml): Performed @RT

Complex I: in 5mM Tris-HCl 7.4 with 50ul of 10mg/ml NADH and 25 mg of NBT (Nitro Blue Tetrazolium) for 5-10min.

Complex II: in 5 mM Tris-HCl 7.4 with 200 ul of 1M Na Succinate, 8ul of phenazine methosulfate (250mM in DMSO) and 25mg NBT for 10-30min.

Complex III: RT in 50mM Na Phosphate 7.2 with 5mg Diaminobenzadine for 1h.

Complex IV: RT in 50mM Na Phosphate 7.2 with 5mg Diaminobenzadine and 100ul of 5mM horse heart cytochrome c for 30min.

Complex V: Incubate the gel in 35 mM Tris/270mM Glycine 8.3 for 2h. Then shift it into the same buffer with 14mM MgSO? 4, 0.2% Pb(NO 3)2, 8mM ATP until a white precipitate appears at the enzyme’s band.

Cathode Buffer/Detergent combinations for individual ox-phos complexes:

Complex I: No Dye or Detergent, CBB, 0.02% DDM / 0.05%DOC (0.05%TX100/0.05%DOC shows intermediates)

Complex II: 0.02%DDM/0.05DOC

Complex III: No Dye or Detergent, 0.02% DDM / 0.05%DOC

Complex IV: 0.05%TX100/0.05%DOC

Complex V: 0.05%TX100/0.05%DOC

Western Blotting:

Western blotting is done like any other protein blotting. Transfer buffer has 0.05% SDS and the transfer is typically performed at 25V for 2H in a cold room. CBB dye on the blot can be washed with MeOH? for 15-20 min. After this step wash the blot with ddH 2 0 twice, wash with TBST once and go for blocking and antibody incubations.

2-D:

The Gel (normally 1.5 mm thick) strip from the native gel is incubated in the 1X SDS-PAGE sample buffer for about 30 minutes. Its then squeezed between the two gel plates, with 0.75 mm spacers, at about the place where we have the wells in a normal gel. Cast the 12% acrylamide-tricene mix for the resolving gel and let it polymerize. A 5% acrylamide stacking mix is poured till 0.5-1 cm away from the bottom of the native gel strip. Once it is polymerized fill the rest of gel and the gaps between the native gel strip and the spacers with the 4% acrylamide mix used for the native gel. You may like to have some wells for loading markers etc. Use a comb with a couple of teeth on a side for this purpose. Run the gel at 90-100 volts in a cold room until the dye front reaches end of the gel.

* Adapted from: Wittig I, Karas M, Sch\xE4gger H., Mol Cell Proteomics.? 6:1215-25 (2007) except for the Bis-Tris buffering.

Topic revision: r1 - 12 Feb 2016, MartyBrandon
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