ska at icbr.ifas.ufl.edu
Mon Mar 17 17:34:12 EST 1997
KLAUS LEHNERT wrote:
> Hi netter's
> does anyone out there have a good (and tried) protocol for
> Tris-Tricine Polyacrylamide gels?
> I have tried the protocols in the "original" publication
> (Schaeger+Jagow 1987), I'm using high-quality reagents, but at
> the very best I'm getting very fuzzy bands, and even when I run
> the gel (in a BioRad Mini-ProteanII) for twice as long (i.e.
> duoble the time the bluemarker needs to run out), my smallest
> marker band (Aprotinin, 6.5kDa) is still in the upper half of
> the gel.
> Any suggestions?
I have routinely used tricine gels for analyzing my small 14 kDa
protein. I use the following recipes:
Separating Gel: (15%T, 6%C)
BioRad's minigels, 0.5 mm spacers, two gels.
12 ml total volume
1.9 ml distilled water
4 ml gel buffer
6 ml 30% polyacrylamide, Bis stock
60 ul 20%SDS
5 ul TEMED
50 ul 10% APS (Ammonium persulfate)
2 gels, BioRad's miniprotean, 0.5 mm spacers
3.18 ml distilled water
1.24 ml gel buffer
0.5 ml 30% polyacrylamide, Bis stock
25 ul 20%SDS
5 ul TEMED
50 ul 10% APS
5 ml total volume.
3M Tris, pH 8.45
(72.6 g Tris, 3ml of 20%SDS, in 200 ml final volume)
Fill up the top buffer tank with 1X Cathode buffer
10 X Cathode Buffer:
1 M Tris, pH 8.25 (Do not adjust pH)
1 M Tricine
(60.5 g Tris, 90 g Tricine, 25 ml of 20% SDS stock, final volume 500 ml)
Dilute 20 ml of 10 X Cathode Buffer to 200 ml with water to make 1 X
Fill up the lower tank with 1 X Anode buffer.
10 X Anode buffer:
2.0 M Tris, pH 8.9
(121 g Tris in 500 ml final volume)
Dilute 40 ml of 10 X Anode Buffer to 400 ml final volume with water to
get 1 X Anode Buffer.
Keep your buffer stocks in fridge. Do not reuse buffers.Dilute your
cathode and Anode buffers just before use.
Run your gels at 50 mA constant current. The bromophenol blue dye should
reach the bottom of the gels in about 3 hours.
Try this protocol. Let me know if you run into any problems. Good luck.
More information about the Methods