BLUE NATIVE GELS

Solutions

10X Sample Buffer 5% Coomassie Briliant Blue g-250 (Serva), 0.5 M 6-aminocaproic acid, 100 mM Bistris/HCl pH 7.0, 1 mM PMSF (optional)
(alternative for stronger detergent concentration: 0.75 M 6-aminocaproic acid, 50 mM Bistris, can also use Serva Blue G)

detergent solution depending on application (see below)

Cathode Buffer 50 mM Tricine, 15 mM BisTris/HCl pH 7.0, 0.02% Coomassie Blue G-250 (Serva)

Colourless Cathode Buffer as above without Coomassie

Anode Buffer 50 mM BisTris/HCl pH 7.0

Gel Buffer 100 mM BisTris/HCl pH 7.0, 1 M 6-aminocaproic acid

Acrylamide solution 48% acrylamide, 1.5% bisacrylamide (make sure it is in solution)

Molecular Weight Markers:
HMW Electrophoresis calibration Kit Pharmacia. Resuspend content of one vial in 250 µl of buffer: 0.16% dodecylmaltoside (detergent chosen depends on application) 10% glycerin, 20 mM Hepes ph 7.4, 50 mM NaCl, + 25 µl sample buffer

Stock solutions 1M Tricine pH 7.0 (adjusted with NaOH) kept at 4CM 1M BisTris/HCl pH 7.0 kept at 4C 2% Serva G Blue in H20

Gels 6-16% Gradient

For 4 X 1.5 mm gels (13cmX12 cm)

We usually use a 12 well com.

For complexes of 300 and greater, 5 -12% gradient is good for resolution

	6%	16%	Stacking

H2O	26.25 ml	----	15.5 ml
Gel Buffer	35 ml	30 ml	18.75 ml
Acrylamide solution	8.75 ml	20 ml	3.0 ml
87% glycerin stock	-----	10 ml	-----

__________________________________________________

	70 ml		60 ml	37.25 ml

For 1 gel:	12.5 ml		12.5 ml
	5 µl	TEMED	5 µl
	50 µl	10% APS	50 µl

Stacker: For 7.5 mls, 6 µl TEMED

60 µl 10% APS

Gels can be stored for several weeks at 4 C when wrapped well.

Sample preparation/Migration of gel

For mitos, we run up to 400 µg total solubilized protein in 160 µl per lane (with 16 µl SB). Adjust protocol for different membrane complexes.
Migration is ON/4C, 100 Volts constant (that gives about 15 mA current). Next day, change the cathode buffer to colourless buffer, increase to 400 Volts let migrate until Blue is out. (Changing decreases amount of Blue in transfer and helps for immunoblot analysis. Alternatively, if proteins precipitate, then do not change blue or just decrease concentration to 0.002%)

Alternative: run fast at 400 V (takes about 4-5 hours), some complexes may fall apart though. Change of cathode to colourless buffer helps transfer to PVDF membrane for Western blotting. However,if you run the gel from the beginning using colourless cathode buffer, resolution is decreased.

After the Migration

A. To stain proteins in the gel:

Put gel in destaining solution ( 25% MeOH, 10% Acetic acid), shaker, RT 1-2 hrs up to ON.

B. Western Blotting

Equilibrate gel in colourless cathode buffer 15 min, RT to remove as much as possible excess Coomassie blue.
Equilibrate PVDF membrane in 100% MeOH at least 5 min, RT
Blotting buffer, Tris-Glycine, with 20% MeOH (low concentrations of up to SDS 0.05% may help transfer of hydrophobic proteins) for wet transfer.
Blot for 2 hrs/4C
For dry transfer, a uniform buffer is used. Wet PVDF with methanol and soak in electrode buffer (50 mM Tricine, 15 mM Bistris, pH 7.0). Place gel and PVDF membranes between 3 mm stacks of papers soaked with electrode buffer. Place a 5 kg load on top, before transfer is started, allow transfer to occur at 20 V for 1 hr, destain to reduce background in 25% methanol, 10% acetic acid ( perhaps destaining is not necessary)
After blotting, proceed as usual for antibody incubation etc.To visualize Mol. weight markers, destain lane in 25% MeOH/10% Ac. acid.
For autoradiography, the blue native gel is stained with Coomassie brilliant blue R250, destained and dried before placing into casette

Solubilization of mitos

For mitos, we often use 1% digitonin (Fluka) or 1.6 mg/ml n-dodecylmaltoside (Fluka) and solublilize at 2.5 mg/ml mito concentration, but this must be optimized.

Buffer contains 50 mM NaCl, 10% glycerol, 20 mM Hepes KOH, pH 7.4, 2.5 mM Mg2Cl, 1 mM EDTA, 1mM BME, PMSF and protease inhibitors.

Solubilize on ice with gentle pipetting and inversion occasionally for 30 min, spin in Ultra at 100,000g for 10 min to remove insoluble material

For one dimension BN gels, you need clean antibodies. If not clean, make a 2D gel. For second dimension run 16% or 10% Tris-Tricine gel (see below)

Denaturing Second Dimension

Cut out pieces or lane from the first (BN) gel, extra lanes can be stored at -20C. Soak gel piece in 1% SDS with 10 mM BME for 15 min -30 min, change to 1% SDS for another 15-30 min. Place strip on top of glass plate at the usual position for the wells. Cut off blue blob of stain at bottom, it is a pain in e second dimension. You may want to make a well on each side using the bottom of a spacer or a cut comb. Std and solubilized mitos can be loaded in these lanes.
Remove excess SDS as completely as possible
Position spacers (now use 0.7 mm thick, when pieces from 1.6 mm native gels are used) and put the second glass plate on top. The 1.6 mm native gels are thereby squeezed and do not slide down when the gel is brought to a vertical position.
Pour separating gel using Tris-Tricine or your favorite gel system, leave a 2 cm gap below gel slide and 0verlay with your favorite solution to promote polymerization.
Cover separating gel with stacking gel after polymerization of separating gel. Overlay with your favorite solution to promote polymerization.
Finally fill the space between the plates, to fully embed the pieces of first dimension gel with the following solution:

For 12 mls
3.0 ml	50% glycerol
240µl	10% SDS
1.0 ml	48% acrylamide (same as used for BN gels)
6.0 ml	BN Gel Buffer (2X)
1.76 ml H20

For polymerization, TEMED 10 µl, 10% APS 100 µl

Allow polymerization to complete for 30 min

7. Run second gel as usual.

Tricine Gels

Schägger and von Jagow 1987. Analytical Biochemistry 166, 368-379.

	16.5/3%	10/3%	Stacking

30% Acryl. 0.8% bisacryl.	16 ml	9.6 ml	1.5 ml
2.2% Bisacryl.	1 ml	0.6 ml	0.06
3M Tris/Cl 0.3% SDS pH 8.45	10 ml	10 ml	3.1 ml
87 % Glycerol	3 ml	3 ml	-
2% polymerised acrylamide	(4 ml)	(4 ml)	-
Water	-	7 ml (3 ml)	7.9 ml

TEMED	20 µl	20 µl	10 µl
10% APS	200 µl	200 µl	100 µl

Volumes are for two 0.8 x 155 x 180 mm gels (thin / long).

Polymerise separating and spacer gels together, not sequentially.

The stacking gel can be added later.

16 % gels run for approx. 220 mAmp h, 10 % gels for 180 mAmp h; max. 45 mAmp

Gel buffer: 3 M Tris - HCl pH 8.45, 0.3 % SDS

Anode buffer: 0.2 M Tris - HCl pH 8.9

Cathode buffer: 0.1 M Tris, 0.1 M Tricine, 0.1 % SDS; pH approx. 8.25 - Do not adjust

Sample buffer: 4 % SDS, 12 % glycerol (w/v), 50 mM Tris, 2 % b mercapto-ethanol (v/v), 0.01% Serva Blue G, adjust to pH 6 with HCl.

2% polymerised linear acrylamide mw 5,000,000 (Polysiences) in water

References

Dekker, P. J., Falk,M., Maarse,A.C., Bomer,U., Muller,H., Guiard,B., Meijer,M., Rassow,J., and Pfanner,N. 1997. The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70–Tim44, EMBO J.,16, 5408-5419

Dekker,P.J., Müller,H., Rassow,J. and Pfanner,N. (1996) Characterization of the preprotein translocase of the outer mitochondrial membrane by blue native electrophoresis. Biol. Chem., 377, 535-8.

Schägger,H., Cramer,W.A. and von Jagow,G. (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem., 217, 220-30.

Schägger,H. and von Jagow,G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem., 166, 368-79.

Schägger,H. and von Jagow,G. (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem., 199, 223-31.